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.

In the vertebrate immune system, each B-lymphocyte expresses a surface IgM-class B cell receptor (BCR). When cross-linked by antigen or anti-IgM antibody, the BCR accumulates with other proteins into distinct surface clusters that activate cell signaling, division, or apoptosis. However, the molecular composition of these clusters is not well defined. Here we describe a quantitative assay we call selective proteomic proximity labeling using tyramide (SPPLAT). It allows proteins in the immediate vicinity of a target to be selectively biotinylated, and hence isolated for mass spectrometry analysis. Using the chicken B cell line DT40 as a model, we use SPPLAT to provide the first proteomic analysis of any BCR cluster using proximity labeling. We detect known components of the BCR cluster, including integrins, together with proteins not previously thought to be BCR-associated. In particular, we identify the chicken B-lymphocyte allotypic marker chB6. We show that chB6 moves to within about 30–40 nm of the BCR following BCR cross-linking, and we show that cross-linking chB6 activates cell binding to integrin substrates laminin and gelatin. Our work provides new insights into the nature and composition of the BCR cluster, and confirms SPPLAT as a useful research tool in molecular and cellular proteomics.

Within cells, proteins can co-assemble into functionally integrated and spatially restricted multicomponent complexes. Often, the affinities between individual proteins are relatively weak, and proteins within such clusters may interact only indirectly with many of their other protein neighbors. This makes proteomic characterization difficult using methods such as immunoprecipitation or cross-linking. Recently, several groups have described the use of enzyme-catalyzed proximity labeling reagents that covalently tag the neighbors of a targeted protein with a small molecule such as fluorescein or biotin. The modified proteins can then be isolated by standard pulldown methods and identified by mass spectrometry. Here we will describe the techniques as well as their similarities and differences. We discuss their applications both to study protein assemblies and to provide a new way for characterizing organelle proteomes. We stress the importance of proteomic quantitation and independent target validation in such experiments. Furthermore, we suggest that there are biophysical and cell-biological principles that dictate the appropriateness of enzyme-catalyzed proximity labeling methods to address particular biological questions of interest.

Background: The microtubule associated protein tau is the principle component of neurofibrillar tangles, which are a characteristic marker in the pathology of Alzheimer's disease; similar lesions are also observed after chronic alcohol abuse. Formaldehyde is a common environmental contaminant and also a metabolite of methanol. Although many studies have been done on methanol and formaldehyde intoxication, none of these address the contribution of protein misfolding to the pathological mechanism, in particular the effect of formaldehyde on protein conformation and polymerization.Results: We found that unlike the typical globular protein BSA, the natively-unfolded structure of human neuronal tau was induced to misfold and aggregate in the presence of ~0.01% formaldehyde, leading to formation of amyloid-like deposits that appeared as densely staining granules by electron microscopy and atomic force microscopy, and bound the amyloid-specific dyes thioflavin T and Congo Red. The amyloid-like aggregates of tau were found to induce apoptosis in the neurotypic cell line SH-SY5Y and in rat hippocampal cells, as observed by Hoechst 33258 staining, assay of caspase-3 activity, and flow cytometry using Annexin V and Propidium Iodide staining. Further experiments showed that Congo Red specifically attenuated the caspase-3 activity induced by amyloid-like deposits of tau.Conclusion: The results suggest that low concentrations of formaldehyde can induce human tau protein to form neurotoxic aggregates, which could play a role in the induction of tauopathies.