Tau N-Terminal Inserts Regulate Tau Liquid-Liquid Phase Separation and Condensates Maturation in a Neuronal Cell Model.

The microtubule-associated protein tau can undergo liquid-liquid phase separation (LLPS) to form membraneless condensates in neurons, yet the underlying molecular mechanisms and functions of tau LLPS and tau droplets remain to be elucidated. The human brain contains mainly 6 tau isoforms with different numbers of microtubule-binding repeats (3R, 4R) and N-terminal inserts (0N, 1N, 2N). However, little is known about the role of N-terminal inserts. Here we observed the dynamics of three tau isoforms with different N-terminal inserts in live neuronal cell line HT22. We validated tau LLPS in cytoplasm and found that 2N-tau forms liquid-like, hollow-shell droplets. Tau condensates became smaller in 1N-tau comparing with 2N-tau, while no obvious tau accumulated dots were shown in 0N-tau. The absence of N-terminal inserts significantly affected condensate colocalization of tau and p62. The results reveal insights into the tau LLPS assembly mechanism and functional effects of N-terminal inserts in tau.

Fisetin inhibits tau aggregation by interacting with the protein and preventing the formation of ß-strands.

Alzheimer's disease is a neurodegenerative disease which severely impacts the health of the elderly. Current treatments are only able to alleviate symptoms, but not prevent or cure the disease. The neurofibrillary tangles formed by tau protein aggregation are one of the defining characteristics of Alzheimer's disease, so tau protein has become a key target for the drug design. In this study, we show that fisetin, a plant-derived polyphenol compound, can inhibit aggregation of the tau fragment, K18, and can disaggregate tau K18 filaments in vitro. Meanwhile it is able to prevent the formation of tau aggregates in cells. Both experimental and computational studies indicate that fisetin could directly interact with tau K18 protein. The binding is mainly created by hydrogen bond and van der Waal force, prevents the formation of ß-strands at the two hexapeptide motifs, and does not perturb the secondary structure or the tubulin binding ability of tau protein. In summary, fisetin might be a candidate for further development as a potential preventive or therapeutic drug for Alzheimer's disease.

Functional characterization of an unknown soybean intrinsically disordered protein in vitro and in Escherichia coli.

Intrinsically disordered proteins (IDPs) possess a wide range of biological function in all organisms, however the specific functions of most IDPs are still unknown. Soybean LOC protein, LOC for short, is a heat-stable protein, which is more abundant in the stress-resistant radicles. Sequence alignment and phylogenetic analysis showed that LOC is a functionally unknown protein and conserved in Fabaceae. LOC, being enriched in most disorder-promoting residues and depleted in most order-promoting residues, was predicted to contain high levels of intrinsic disorder by several commonly used computational tools. However, it was also predicted to contain two disorder-based protein-protein binding sites and two short α-helical segments. The circular dichroism spectroscopic analysis showed that this protein is mostly disordered in water, but can form more α-helical structure in the presence of SDS and TFE. Functional in vitro studies showed that the LOC protein is able to prevent lactate dehydrogenase inactivation by freeze-thaw at a molar ratio of 10:1. Furthermore, in vivo analyses revealed the survival rate of Escherichia coli over-expressing LOC protein under the conditions of osmotic stress was noticeably increased in comparison with the control. These observations suggest that the intrinsically disordered protein LOC might serve as a chaperone and/or cell protector.