TY  - THES
AU  - Chinnathambi, Subash
TI  - Influence of Phosphorylation on the Pathological Conformation of Human Microtubule Associated Protein Tau
PB  - Universität Hamburg
VL  - Dissertation
M1  - DZNE-2024-00741
SP  - 159 pages
PY  - 2011
N1  - Dissertation, Universität Hamburg, 2011
AB  - Tau is one of the microtubule associated proteins which can bind and stabilize microtubules. It is classified as a natively unfolded protein. It has a very low content of secondary structural elements. Tau aggregates into insoluble fibers called paired helical filaments in Alzheimer’s disease. The residual folding and intramolecular interactions within tau are largely unknown, and the factors contributing to the formation and stability of PHFs and different morphologies of PHFs are not well understood.The results of the present studies are aimed at better understanding of the effect of phosphorylation on the aggregation propensity and tau microtubule interaction.The effect of phosphorylation sites in the KXGS motifs within the repeats and in the SP/TP motifs of the flanking regions were studied. We also studied tau fibrilpolymorphism by Electron Microscopy and Atomic Force Microscopy. We further studied tau in solution by increasing temperature using different biophysical methods (CD spectroscopy, static and dynamic light scattering and small angle x-ray scattering). These observations could form a basis for elucidating the molecular and physiological role of phosphorylation at the repeat domains and in the flanking regions. The results are summarized as follows(i) The pseudo-phosphorylation of KXGE motifs within the repeat domains of tau has a strong negative effect on both the tau-microtubule interaction and the aggregation into paired helical filaments. KXGE mutants on the basis of three repeat domain and four repeat domain constructs show a low tendency to aggregate and form amorphous aggregates instead of PHFs. Pseudo-phosphorylation in the SP/TP motifs in the N- and C-terminal domains of the flanking regions has only a moderate effect, both on the tau-microtubule interaction and on the aggregation into paired helical filaments. Overall, the results show that the physiological tau-microtubule interaction and the pathological tau-tau interaction similarly can be influenced by phosphorylation.(ii) The combined pseudo-phosphorylation of AT8* and PHF1 sites leads to compaction of the paperclip conformation, such that the N-terminus approaches the repeat domain. When the Cterminus tail is cleaved from tau, the compaction becomes even stronger by combining pseudophosphorylated AT8*, AT100, and PHF1 epitopes. This is accompanied by a strong increase in the reaction with conformation-dependent antibodies MC1 and Alz-50, suggesting the generation of a pathological conformation characteristic of tau in AD. Furthermore, the compact paperclip conformation enhances the aggregation propensity to paired helical filaments but has little effect on microtubule interactions. Discussion 89(iii) High resolution atomic force and electron microscopy were employed to study fibrilsassembled from different human tau isoforms and domains. All fibrils reveal structuralpolymorphism: the “thin twisted” and “thin smooth” fibrils resemble flat ribbons(cross-section  10 x 15 nm) with diverse twist periodicities. “Thick fibrils” show periodicities of  65-70 nm and thicknesses of  9-18 nm as routinely reported for“paired helical filaments” but structurally resemble heavily twisted ribbons.Therefore, thin and thick fibrils assembled from different human tau isoforms challenge current structural models of paired helical filaments. The full-length tau and repeat domain constructs assemble into fibrils of similar thickness, the “fuzzy coat” of tau protein termini surrounding the fibril axis is nearly invisible for atomicforce microscopy and electron microscopy, presumably because of its high flexibility.(iv) The unfolded nature of tau protein allows it to adopt either more extended or compact conformations. It is intriguing to speculate that the compaction and memory effect observed after rapid heating/cooling may be related to the compaction observed upon hyperphosphorylation, characteristic of incipient neuronal degeneration in AD. The “memory effect” is observed with full-length tau, but not with the repeat domain alone, suggesting that the interplay between domains in the whole protein might be responsible for the effect. One possible explanation could be the interplay between the acidic N-terminal domain (which varies among the tau isoforms due to alternative splicing) and the basic repeat domain (which also differs between the isoforms).
LB  - PUB:(DE-HGF)11
UR  - https://pub.dzne.de/record/270141
ER  -