Potnuru Lokeswara Rao, DuBose Austin, Nowotarski Mesopotamia S, Vigers Michael, Zhang Boqin, Han Chung-Ta, Han Songi
Department of Chemistry, Northwestern University, Evanston 60208 Illinois, United States of America.
Department of Chemistry and Biochemistry, University of California Santa Barbara, California 93106 United States of America.
bioRxiv. 2024 Aug 17:2024.08.14.606685. doi: 10.1101/2024.08.14.606685.
Hyperphosphorylation of the protein tau is one of the biomarkers of neurodegenerative diseases in the category of tauopathies. However, the molecular level, mechanistic, role of this common post-translational modification (PTM) in enhancing or reducing the aggregation propensity of tau is unclear, especially considering that combinatorial phosphorylation of multiple sites can have complex, non-additive, effects on tau protein aggregation. Since tau proteins stack in register and parallel to elongate into pathological fibrils, phosphoryl groups from adjacent tau strands with 4.8 Å separation must find an energetically favorable spatial arrangement. At first glance, this appears to be an unfavorable configuration due to the proximity of negative charges between phosphate groups from adjacent neighboring tau fibrils. However, this study tests a counterhypothesis that phosphoryl groups within the fibril core-forming segments favorably assemble into highly ordered, hydrogen-bonded, one-dimensionally extended wires under biologically relevant conditions. We selected two phosphorylation sites associated with neurodegeneration, serine 305 (S305) and tyrosine 310 (Y310), on a model tau peptide jR2R3-P301L (tau295-313) spanning the R2/R3 splice junction of tau, that readily aggregate into a fibril with characteristics of a seed-competent mini prion. Using multiple quantum spin counting (MQ-SC) by P solid-state NMR of phosphorylated jR2R3-P301L tau peptide fibrils, enhanced by dynamic nuclear polarization, we find that at least six phosphorous spins must neatly arrange in 1D within fibrils or in 2D within a protofibril to yield the experimentally observed MQ-coherence orders of four. We found that S305 stabilizes the tau fibrils and leads to more seeding-competent fibrils compared to jR2R3 P301L or Y310. This study introduces a new concept that phosphorylation of residues within a core forming tau segment can mechanically facilitate fibril registry and stability due a hitherto unrecognized role of phosphoryl groups to form highly ordered, extended, 1D wires that stabilize pathological tau fibrils.
蛋白质tau的过度磷酸化是tau蛋白病类神经退行性疾病的生物标志物之一。然而，这种常见的翻译后修饰（PTM）在增强或降低tau聚集倾向方面的分子水平、机制和作用尚不清楚，尤其是考虑到多个位点的组合磷酸化对tau蛋白聚集可能产生复杂的、非加性的影响。由于tau蛋白沿直线堆积并平行排列以延伸形成病理性纤维，相邻tau链上相距4.8 Å的磷酸基团必须找到能量上有利的空间排列。乍一看，由于相邻tau纤维上磷酸基团之间负电荷的接近，这似乎是一种不利的构型。然而，本研究检验了一种反假设，即在生物学相关条件下，纤维核心形成段内的磷酸基团有利于组装成高度有序的、氢键连接的一维延伸链。我们在跨越tau的R2/R3剪接连接点的模型tau肽jR2R3-P301L（tau295-313）上选择了两个与神经退行性变相关的磷酸化位点，丝氨酸305（S305）和酪氨酸310（Y310），该肽很容易聚集形成具有种子样活性的微型朊病毒特征的纤维。通过动态核极化增强的31P固体核磁共振对磷酸化的jR2R3-P301L tau肽纤维进行多量子自旋计数（MQ-SC），我们发现至少六个磷自旋必须在纤维内一维排列或在原纤维内二维排列，才能产生实验观察到的MQ相干阶数为四。我们发现，与jR2R3 P301L或Y310相比，S305能稳定tau纤维并导致更多具有种子样活性的纤维。本研究引入了一个新概念，即核心形成tau段内残基的磷酸化可以通过磷酸基团形成高度有序的、延伸的一维链来机械地促进纤维排列和稳定性，而这种作用迄今未被认识到，这些一维链可稳定病理性tau纤维。
