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.

The microtubule associated protein tau (MAPT/tau) aberrantly accumulates in fifteen neurodegenerative diseases, termed tauopathies. One way to treat tauopathies may be to accelerate tau clearance, but the molecular mechanisms governing tau stability are not yet clear. We recently identified chemical probes that markedly accelerate the clearance of tau in cellular and animal models. In the current study, we used one of these probes in combination with immunoprecipitation and mass spectrometry to identify 48 proteins whose association with tau changes during the first 10 minutes after treatment. These proteins included known modifiers of tau proteotoxicity, such as ILF-2 (NFAT), ILF-3, and ataxin-2. A striking observation from the dataset was that tau binding to heat shock protein 70 (Hsp70) decreased while binding to Hsp90 significantly increased. Both chaperones have been linked to tau homeostasis, but their mechanisms have not been established. Using peptide arrays and binding assays, we found that Hsp70 and Hsp90 appeared to compete for binding to shared sites on tau. Further, the Hsp90-bound complex proved to be important in initiating tau clearance in cells. These results suggest that the relative levels of Hsp70 and Hsp90 may help determine whether tau is retained or degraded. Consistent with this model, analysis of reported microarray expression data from Alzheimer’s disease patients and age-matched controls showed that the levels of Hsp90 are reduced in the diseased hippocampus. These studies suggest that Hsp70 and Hsp90 work together to coordinate tau homeostasis.

Heat shock protein 70 (Hsp70) plays critical roles in proteostasis and is an emerging target for multiple diseases. However, competitive inhibition of the enzymatic activity of Hsp70 has proven challenging and, in some cases, may not be the most productive way to redirect Hsp70 function. Another approach is to inhibit Hsp70’s interactions with important co-chaperones, such as J proteins, nucleotide exchange factors (NEFs) and tetratricopeptide repeat (TPR) domain-containing proteins. These co-chaperones normally bind Hsp70 and guide its many diverse cellular activities. Complexes between Hsp70 and co-chaperones have been shown to have specific functions, such as pro-folding, pro-degradation and pro-trafficking. Thus, a promising strategy may be to block protein-protein interactions between Hsp70 and its co-chaperones or to target allosteric sites that disrupt these contacts. Such an approach might shift the balance of Hsp70 complexes and re-shape the proteome and it has the potential to restore healthy proteostasis. In this review, we discuss specific challenges and opportunities related to those goals. By pursuing Hsp70 complexes as drug targets, we might not only develop new leads for therapeutic development, but also discover new chemical probes for use in understanding Hsp70 biology.

Nitriles have been shown to be effective vibrational probes of local environments in proteins but have yet to be fully utilized for the study of nucleic acids. The potential utility of 5-cyano-2'-deoxyuridine ( 1) as a probe of local nucleic acid environment was investigated by measuring the dependence of the IR nitrile stretching frequency (nu CN), line shape, and absorbance on solvent and temperature. The nu CN was found to be sensitive to solvent with an observed blue shift of 9.2 cm (-1) in going from THF to water. The dependence of the nitrile IR absorbance band was further investigated in water-THF mixtures. Global line shape analysis, difference FTIR spectroscopy, and singular value decomposition (SVD) were used to show the presence of three distinct local environments around the nitrile group of 1 in these mixtures. A modest blue shift in nu CN was observed upon a hydrogen-bond-mediated heterodimer formation between 2 (a silyl ether analogue of 1) and 2,6-diheptanamido-pyridine ( 3a) in chloroform. The intrinsic temperature dependence of the nu CN was found to be minimal and linear over the temperature range studied. The experimental studies were complemented by density functional theory (DFT) calculations on the dependence of the nitrile stretching frequency on solute-solvent interactions and upon heterodimer formation with model systems.