Liquid–liquid phase separation (LLPS) of proteins into biomolecular condensates has emerged as a fundamental principle underpinning cellular function and malfunction. Indeed, many human pathologies, including protein misfolding diseases, are linked to aberrant liquid-to-solid phase transitions, and disease-associated protein aggregates often nucleate through phase separation. The molecular level determinants that promote pathological phase transitions remain, however, poorly understood. Here we study LLPS of the microtubule-associated protein Tau, whose aberrant aggregation is associated with a number of neurodegenerative diseases, including Alzheimer’s disease. Using single molecule spectroscopy, we probe directly the conformational changes that the protein undergoes as a result of LLPS. We perform single-molecule FRET and fluorescence correlation spectroscopy experiments to monitor the intra- and intermolecular changes and demonstrate that the N- and C-terminal regions of Tau become extended, thus exposing the microtubule-binding region. These changes facilitate intermolecular interactions and allow for the formation of nanoscale clusters of Tau. Our results suggest that these clusters can promote the fibrillization of Tau, which can be dramatically accelerated by disease-related mutations P301L and P301S. Our findings thus provide important molecular insights into the mechanism of protein phase separation and the conversion of protein condensates from functional liquid assemblies to pathological aggregates.

Significance: Protein oligomers are a fundamental component of amyloidogenic disorders such as Alzheimer’s disease, being both critical intermediates in the formation of amyloid fibrils and the most toxic species on the protein aggregation pathway. Here, we derive general analytical expressions for the chemical kinetics of the oligomers generated during amyloid-formation reactions and explain how these may be used to categorize oligomers according to their basic physicochemical properties. By applying this framework to experimental kinetic data on the aggregation of a wide range of amyloidogenic proteins, we are able to determine features common to all amyloid oligomers. In particular, most are not capable of rapid growth by monomer addition, and most dissociate rather than mature into fibrils.

Cellular redox homeostasis is precisely balanced by generation and elimination of reactive oxygen species (ROS). ROS are not only capable of causing oxidation of proteins, lipids and DNA to damage cells but can also act as signaling molecules to modulate transcription factors and epigenetic pathways that determine cell survival and death. Hsp70 proteins are central hubs for proteostasis and are important factors to ameliorate damage from different kinds of stress including oxidative stress. Hsp70 members often participate in different cellular signaling pathways via their clients and cochaperones. ROS can directly cause oxidative cysteine modifications of Hsp70 members to alter their structure and chaperone activity, resulting in changes in the interactions between Hsp70 and their clients or cochaperones, which can then transfer redox signals to Hsp70-related signaling pathways. On the other hand, ROS also activate some redox-related signaling pathways to indirectly modulate Hsp70 activity and expression. Post-translational modifications including phosphorylation together with elevated Hsp70 expression can expand the capacity of Hsp70 to deal with ROS-damaged proteins and support antioxidant enzymes. Knowledge about the response and role of Hsp70 in redox homeostasis will facilitate our understanding of the cellular knock-on effects of inhibitors targeting Hsp70 and the mechanisms of redox-related diseases and aging.

Preventing tau aggregation is a potential therapeutic strategy in Alzheimer’s disease and other tauopathies. Recently, liquid–liquid phase separation has been found to facilitate the formation of pathogenic tau conformations and fibrillar aggregates, although many aspects of the conformational transitions of tau during the phase transition process remain unknown. Here, we demonstrate that the tau aggregation inhibitor methylene blue promotes tau liquid–liquid phase separation and accelerates the liquid-to-gel transition of tau droplets independent of the redox activity of methylene blue. We further show that methylene blue inhibits the conversion of tau droplets into fibrils and reduces the cytotoxicity of tau aggregates. Although gelation slows down the mobility of tau and tubulin, it does not impair microtubule assembly within tau droplets. These findings suggest that methylene blue inhibits tau amyloid fibrillization and accelerates tau droplet gelation via distinct mechanisms, thus providing insights into the activity of tau aggregation inhibitors in the context of phase transition.

Heat shock protein 70 (Hsp70) proteins are a family of ancient and conserved chaperones. Cysteine modifications have been widely detected among different Hsp70 family members in vivo, but their effects on Hsp70 structure and function are unclear. Here, we treated HeLa cells with diamide, which typically induces disulfide bond formation except in the presence of excess GSH, when glutathionylated cysteines predominate. We show that in these cells, HspA1A (hHsp70) undergoes reversible cysteine modifications, including glutathionylation, potentially at all five cysteine residues. In vitro experiments revealed that modification of cysteines in the nucleotide-binding domain of hHsp70 is prevented by nucleotide binding but that Cys-574 and Cys-603, located in the C-terminal α-helical lid of the substrate-binding domain, can undergo glutathionylation in both the presence and absence of nucleotide. We found that glutathionylation of these cysteine residues results in unfolding of the α-helical lid structure. The unfolded region mimics substrate by binding to and blocking the substrate-binding site, thereby promoting intrinsic ATPase activity and competing with binding of external substrates, including heat shock transcription factor 1 (Hsf1). Thus, post-translational modification can alter the structure and regulate the function of hHsp70.