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. 2012 Oct 19;7(10):1677-86.
doi: 10.1021/cb3002599. Epub 2012 Jul 25.

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

Andrea D Thompson  1 , K Matthew Scaglione, John Prensner, Anne T Gillies, Arul Chinnaiyan, Henry L Paulson, Umesh K Jinwal, Chad A Dickey, Jason E Gestwicki
Affiliations

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

Andrea D Thompson et al. ACS Chem Biol. .

Abstract

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.

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Figures

Figure 1
Figure 1. Proteomic analysis of the proteins associated with tau during the acute switch to a degradation fate in response to methylene blue (MB) treatment
(A) Schematic of the experimental strategy for identifying tau-associated proteins which alter tau stability. (B) Using spectral counting, 456 of co-immunoprecipitated proteins did not change in their association with tau in response to MB treatment. However, 20 proteins decreased their association with tau and 28 preferentially bound. (C) Western blots on freshly immunoprecipitated samples confirmed that MB causes Hsp70 to be released from tau, while Hsp90 binding increases.
Figure 2
Figure 2. Methylene blue (MB)-initiated tau degradation is dependent on Hsp90
Hsp90 siRNA does not cause a significant change in total tau levels in untreated cells, but it attenuates tau clearance in response to MB. Quantifications of band intensities from two independent experiments were performed using Image J. (* Student’s two-tailed unpaired t-test, p-value < 0.05).
Figure 3
Figure 3. Hsp70 and Hsp90 bind discrete and partially overlapping sites on tau, as measured by peptide microarrays
(A) Representative array results using Hsp70 nucleotide binding domain (NBD), Hsp90, and Hsp70 (all 10 μM) are shown. Further, the fluorescence intensities at a wavelength of 532 nm, after background subtraction, for each array spot tested in triplicate are shown. Peptides with intensities 3 standard errors of mean (SEM) above the mean for the total dataset are colored in red. Peptides that bound either Hsp70 NBD or antibody alone are shown in gray. One false-positive spot (array ID: 161) was removed for clarity. (B) Schematic of the Hsp70 and Hsp90 binding sites mapped onto 4R0N tau, highlighting the four microtubule-binding repeats (R1–4).
Figure 4
Figure 4. Hsp70 competes with Hsp90 for binding to tau
(A) Immobilized Hsp70 and Hsp90 bind to 4RON tau. (B) Soluble Hsp70 can inhibit binding of tau to immobilized Hsp90 with an IC50 value of 4 μM. As a control, free Hsp90 also competes with this interaction. (C) Soluble Hsp90 only weakly competes with immobilized Hsp70 for binding to tau with an IC50 value greater than 50 μM. As a control, free Hsp70 inhibits binding with an IC50 of 8 μM. Similar values were seen using different Hsp70 and Hsp90 isoforms (see Table 2). All of the experiments were performed in triplicate. Results are shown as the average and standard error of the mean.
Figure 5
Figure 5. Hsp90 levels are decreased in hippocampal samples from Alzheimer’s disease (AD) patients
Analysis of the (A) GDS810 and (B) GSE5281 datasets reveals a decrease in the mRNA levels of both isoforms of Hsp90 (HSPAA1and HSP90AB1) in AD samples. There were no statistically significant changes in the levels of most the stress-inducible Hsp70s, such as HSPAB1 (C and D), HSPA1B, HSPA2, HSPA4, or HSPA14 (Table 2 and Supplemental Fig 4). However, the levels of Hsc70 (HSPA8) were decreased in AD patient samples (C and D).
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