Mapping interactions with the chaperone network reveals factors that protect against tau aggregation.

A network of molecular chaperones is known to bind proteins ('clients') and balance their folding, function and turnover. However, it is often unclear 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. Here, 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 note, 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.

Epitope analysis following active immunization with tau proteins reveals immunogens implicated in tau pathogenesis.

BACKGROUND: Abnormal tau hyperphosphorylation and its accumulation into intra-neuronal neurofibrillary tangles are linked to neurodegeneration in Alzheimer's disease and similar tauopathies. One strategy to reduce accumulation is through immunization, but the most immunogenic tau epitopes have so far remained unknown. To fill this gap, we immunized mice with recombinant tau to build a map of the most immunogenic tau epitopes. METHODS: Non-transgenic and rTg4510 tau transgenic mice aged 5 months were immunized with either human wild-type tau (Wt, 4R0N) or P301L tau (4R0N). Each protein was formulated in Quil A adjuvant. Sera and splenocytes of vaccinated mice were collected to assess the humoral and cellular immune responses to tau. We employed a peptide array assay to identify the most effective epitopes. Brain histology was utilized to measure the effects of vaccination on tau pathology and inflammation. RESULTS: Humoral immune responses following immunization demonstrated robust antibody titers (up to 1:80,000 endpoint titers) to each tau species in both mice models. The number of IFN-γ producing T cells and their proliferation were also increased in splenocytes from immunized mice, indicating an increased cellular immune response, and tau levels and neuroinflammation were both reduced. We identified five immunogenic motifs within either the N-terminal (9-15 and 21-27 amino acids), proline rich (168-174 and 220-228 amino acids), or the C-terminal regions (427-438 amino acids) of the wild-type and P301L tau protein sequence. CONCLUSIONS: Our study identifies five previously unknown immunogenic motifs of wild-type and mutated (P301L) tau protein. Immunization with both proteins resulted in reduced tau pathology and neuroinflammation in a tau transgenic model, supporting the efficacy of tau immunotherapy in tauopathy.

Hsp70 protein complexes as drug targets.

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, including roles in pro-folding, pro-degradation and pro-trafficking pathways. 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 these 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.

Synthetic lethal interactions in yeast reveal functional roles of J protein co-chaperones.

J proteins are a diverse family of co-chaperones that cooperate with heat shock protein 70 (Hsp70) to coordinate protein quality control, especially in response to cellular stress. Current models suggest that individual J proteins might play roles in recruiting Hsp70s to specific functions, such as maintaining cell wall integrity or promoting ribosome biogenesis. However, relatively few stresses have been used to test this model and, as a result, only a few specific activities have been identified. To expand our understanding of the J protein network, we used a synthetic lethal approach in which 11 Saccharomyces cerevisiae deletion strains were treated with 12 well-characterized chemical inhibitors. The results defined new roles for specific J proteins in major signaling pathways. For example, an important role for Swa2 in cell wall integrity was identified and activities of the under-explored Jjj1, Apj1, Jjj3 and Caj1 proteins were suggested. More generally, these findings support a model in which some J proteins, such as Ydj1 and Zuo1, play "generalist" roles, while others, such as Apj1 and Jjj2, are "specialists", having roles in relatively few pathways. Together, these results provide new insight into the network of J proteins.

Analysis of the tau-associated proteome reveals that exchange of Hsp70 for Hsp90 is involved in tau degradation.

The microtubule associated protein tau (MAPT/tau) aberrantly accumulates in 15 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 min 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 data set was that tau binding to heat shock protein 70 (Hsp70) decreased, whereas 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.

Molecular chaperones DnaK and DnaJ share predicted binding sites on most proteins in the E. coli proteome.

In Escherichia coli, the molecular chaperones DnaK and DnaJ cooperate to assist the folding of newly synthesized or unfolded polypeptides. DnaK and DnaJ bind to hydrophobic motifs in these proteins and they also bind to each other. Together, this system is thought to be sufficiently versatile to act on the entire proteome, which creates interesting challenges in understanding the interactions between DnaK, DnaJ and their thousands of potential substrates. To address this question, we computationally predicted the number and frequency of DnaK- and DnaJ-binding motifs in the E. coli proteome, guided by free energy-based binding consensus motifs. This analysis revealed that nearly every protein is predicted to contain multiple DnaK- and DnaJ-binding sites, with the DnaJ sites occurring approximately twice as often. Further, we found that an overwhelming majority of the DnaK sites partially or completely overlapped with the DnaJ-binding motifs. It is well known that high concentrations of DnaJ inhibit DnaK-DnaJ-mediated refolding. The observed overlapping binding sites suggest that this phenomenon may be explained by an important balance in the relative stoichiometry of DnaK and DnaJ. To test this idea, we measured the chaperone-assisted folding of two denatured substrates and found that the distribution of predicted DnaK- and DnaJ-binding sites was indeed a good predictor of the optimal stoichiometry required for folding. These studies provide insight into how DnaK and DnaJ might cooperate to maintain global protein homeostasis.

(15)N NMR studies of a nitrile-modified nucleoside.

Nitrile-modified molecules have proven to be excellent probes of local environments in biomolecules via both vibrational and fluorescence spectroscopy. The utility of the nitrile group as a spectroscopic probe has been expanded here to (15)N NMR spectroscopy by selective (15)N incorporation. The (15)N NMR chemical shift (δ((15)N)) of the (15)N-labeled 5-cyano-2'-deoxyuridine (C(15)NdU, 1a) was found to change from 153.47 to 143.80 ppm in going from THF-d(8) to D(2)O. A 0.81 ppm downfield shift was measured upon formation of a hydrogen-bond-mediated heterodimer between 2,6-diheptanamidopyridine and a silyl ether analogue of 1a in chloroform, and the small intrinsic temperature dependence of δ((15)N) of C(15)NdU was measured as a 0.38 ppm downfield shift from 298 to 338 K. The experiments were complemented with density functional theory calculations exploring the effect of solvation on the (15)N NMR chemical shift.

The CHAT project: paediatricians and mental health clinicians: working together for the sake of the children.

INTRODUCTION: The Shared Care model, originally developed to address the paucity of adult mental health resources, has potential applicability to the design and delivery of mental health services for children and youth. With a similar prevalence of mental illness and more difficulty accessing mental health care than adults, Canadian children and youth have been even less well served than their parents. As there is a significant overlap in the types of health concerns for which children and youth are seen by mental health clinicians and paediatricians, building improved collaboration between these groups is an obvious, though under-utilized, method of enhancing mental health care for this population. The CHAT project was a one year collaboration involving three different types of collaborative activities between mental health clinicians and paediatricians. METHODS: Out-patient mental health clinicians and community paediatricians were surveyed about their educational needs and interests. A community paediatrician joined an outpatient mental health care team, continuing professional educational activities were provided by each group for the other and were evaluated. RESULTS: The paediatrician's participation was evaluated positively by team members. Eighty-three percent of mental health clinicians (n=10) rated the educational sessions positively and 77.8% (n=7) noted increased knowledge. Paediatricians expressed strong interest in having more mental health-related educational activities. CONCLUSION: This small project provided opportunities for useful exchanges between mental health clinicians and community paediatricians and preliminary evidence that shared care initiatives for children and youth should be further researched.

A vibrational probe for local nucleic acid environments: 5-cyano-2'-deoxyuridine.

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.