A network of molecular chaperones is known to bind proteins (“clients”) and balance their folding, function and turnover. However, it is often not clear which chaperones are critical for selective recognition of individual clients. It is also not clear why these key chaperones might fail in protein aggregation diseases. In this study, we utilized human microtubule-associated protein tau (MAPT or tau) as a model client to survey interactions between ~30 purified chaperones and ~20 disease-associated tau variants (~600 combinations). From this large-scale analysis, we identified human DnaJA2 as an unexpected, but potent, inhibitor of tau aggregation. DnaJA2 levels were correlated with tau pathology in human brains, supporting the idea that it is an important regulator of tau homeostasis. Of significance, we found that some disease-associated tau variants were relatively immune to interactions with chaperones, suggesting a model in which avoiding physical recognition by chaperone networks may contribute to disease. Impact Statement Large-scale screening of chaperone interactions with tau and its variants identified DnaJA2 as a key protective factor in tauopathy.

Highlights d The FTD-causing V337M tau mutation impairs axon initial segment (AIS) plasticity d The V337M tau mutation impairs activity homeostasis d The V337M tau mutation leads to accumulation of EB3 in the AIS d EB3 is critical for regulating AIS plasticity and activity homeostasis

Molecular chaperones play a central role in protein homeostasis (a.k.a. proteostasis) by balancing protein folding, quality control, and turnover. To perform these diverse tasks, chaperones need the malleability to bind nearly any "client" protein and the fidelity to detect when it is misfolded. Remarkably, these activities are carried out by only ∼180 dedicated chaperones in humans. How do a relatively small number of chaperones maintain cellular and organismal proteostasis for an entire proteome? Furthermore, once a chaperone binds a client, how does it "decide" what to do with it? One clue comes from observations that individual chaperones engage in protein-protein interactions (PPIs)-both with each other and with their clients. These physical links coordinate multiple chaperones into organized, functional complexes and facilitate the "handoff" of clients between them. PPIs also link chaperones and their clients to other cellular pathways, such as those that mediate trafficking (e.g., cytoskeleton) and degradation (e.g., proteasome). The PPIs of the chaperone network have a wide range of affinity values (nanomolar to micromolar) and involve many distinct types of domain modules, such as J domains, zinc fingers, and tetratricopeptide repeats. Many of these motifs have the same binding surfaces on shared partners, such that members of one chaperone class often compete for the same interactions. Somehow, this collection of PPIs draws together chaperone families and creates multiprotein subnetworks that are able to make the "decisions" of protein quality control. The key to understanding chaperone-mediated proteostasis might be to understand how PPIs are regulated. This Account will discuss the efforts of our group and others to map, measure, and chemically perturb the PPIs within the molecular chaperone network. Structural biology methods, including X-ray crystallography, NMR spectroscopy, and electron microscopy, have all played important roles in visualizing the chaperone PPIs. Guided by these efforts and -omics approaches to measure PPIs, new advances in high-throughput chemical screening that are specially designed to account for the challenges of this system have emerged. Indeed, chemical biology has played a particularly important role in this effort, as molecules that either promote or inhibit specific PPIs have proven to be invaluable research probes in cells and animals. In addition, these molecules have provided leads for the potential treatment of protein misfolding diseases. One of the major products of this research field has been the identification of putative PPI drug targets within the chaperone network, which might be used to change chaperone "decisions" and rebalance proteostasis.

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Although the primary protein sequence of ubiquitin (Ub) is extremely stable over evolutionary time, it is highly tolerant to mutation during selection experiments performed in the laboratory. We have proposed that this discrepancy results from the difference between fitness under laboratory culture conditions and the selective pressures in changing environments over evolutionary timescales. Building on our previous work (Mavor et al., 2016), we used deep mutational scanning to determine how twelve new chemicals (3-Amino-1,2,4-triazole, 5-fluorocytosine, Amphotericin B, CaCl2, Cerulenin, Cobalt Acetate, Menadione, Nickel Chloride, p-Fluorophenylalanine, Rapamycin, Tamoxifen, and Tunicamycin) reveal novel mutational sensitivities of ubiquitin residues. Collectively, our experiments have identified eight new sensitizing conditions for Lys63 and uncovered a sensitizing condition for every position in Ub except Ser57 and Gln62. By determining the ubiquitin fitness landscape under different chemical constraints, our work helps to resolve the inconsistencies between deep mutational scanning experiments and sequence conservation over evolutionary timescales.