Understanding the mechanism by which tau binds to and promotes microtubule (MT) assembly as part of its native function may also provide insight into its loss of function that occurs in neurodegenerative disease. Both mechanistic and structural studies of tau have been hindered by its intrinsic disorder and highly dynamic nature. Here, we combine fluorescence correlation spectroscopy and acrylodan fluorescence screening to study the stoichiometry and structural features of tau-tubulin assemblies. Our results show that tau binds to multiple tubulin dimers, even when MT assembly is inhibited. Moreover, we observe helical structure in the repeat regions of the MT binding domain of tau in the tau-tubulin complex, reflecting partial folding upon binding. Our findings support a role for tau’s intrinsic disorder in providing a flexible scaffold for binding tubulin and MTs and a disorder-to-order transition in mediating this important interaction.

Intrinsically disordered protein (IDP) of tau binds and stabilizes microtubule, which contributes to the proper function of neuron, while its aggregation is implicated in Alzheimer's disease. In recent years, we have conducted various extensive molecular dynamics simulations of tau proteins in their monomer, normal fibril, and hyper-phosphorylated filament states. The conformations of two critical hexapeptides (275VQIINK280 and 306VQIVYK311) have changed from random, alpha-helical, and beta-sheets, in concert with overall conformational changes of tau protein. In the isolated monomeric states, we observed the dynamically ordered structures are evolved from disordered conformations. Our REMD simulations revealed the structural diversity of K18 and K19 monomers, including helix-rich and mixed helix-and beta-sheetrich structures. The two VQIXXK motifs have high beta-sheet contents and large hydrophobic surface exposure. The preformed Ab1-42 protofibril can stretch tau conformation, and drastically reduces the metastable secondary structures/hydrogen bonding/salt-bridge networks in tau monomers, and exposes VQIXXK motifs more. In tau amyloid fibril, the VQIXXK motifs can be embedded tightly in linear or bent beta-sheet motifs. However, when N-and C-terminals of tau protein are highly phosphorylated, the VQIXXK motifs in amyloid fibril may have more solvent exposure. References: (1)

Numerous proteins that have hydrophobic transmembrane domains (TMDs) traverse the cytosol and posttranslationally insert into cellular membranes. It is unclear how these hydrophobic membrane proteins evade recognition by the cytosolic protein quality control (PQC), which typically recognizes exposed hydrophobicity in misfolded proteins and marks them for proteasomal degradation by adding ubiquitin chains. Here, we find that tail-anchored (TA) proteins, a vital class of membrane proteins, are recognized by cytosolic PQC and are ubiquitinated as soon as they are synthesized in cells. Surprisingly, the ubiquitinated TA proteins are not routed for proteasomal degradation but instead are handed over to the targeting factor, TRC40, and delivered to the ER for insertion. The ER-associated deubiquitinases, USP20 and USP33, remove ubiquitin chains from TA proteins after their insertion into the ER. Thus, our data suggest that deubiquitinases rescue posttranslationally targeted membrane proteins that are inappropriately ubiquitinated by PQC in the cytosol.

Molecular chaperones in cells constantly monitor and bind to exposed hydrophobicity in newly synthesized proteins and assist them in folding or targeting to cellular membranes for insertion. However, proteins can be misfolded or mistargeted, which often causes hydrophobic amino acids to be exposed to the aqueous cytosol. Again, chaperones recognize exposed hydrophobicity in these proteins to prevent nonspecific interactions and aggregation, which are harmful to cells. The chaperone‐bound misfolded proteins are then decorated with ubiquitin chains denoting them for proteasomal degradation. It remains enigmatic how molecular chaperones can mediate both maturation of nascent proteins and ubiquitination of misfolded proteins solely based on their exposed hydrophobic signals. In this review, we propose a dynamic ubiquitination and deubiquitination model in which ubiquitination of newly synthesized proteins serves as a “fix me” signal for either refolding of soluble proteins or retargeting of membrane proteins with the help of chaperones and deubiquitinases. Such a model would provide additional time for aberrant nascent proteins to fold or route for membrane insertion, thus avoiding excessive protein degradation and saving cellular energy spent on protein synthesis. Also see the video abstract here: https://youtu.be/gkElfmqaKG4

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