Isoform-selective Genetic Inhibition of Constitutive Cytosolic Hsp70 Activity Promotes Client Tau Degradation Using an Altered Co-chaperone Complement

Abstract

The constitutively expressed heat shock protein 70 kDa (Hsc70) is a major chaperone protein responsible for maintaining proteostasis, yet how its structure translates into functional decisions regarding client fate is still unclear. We previously showed that Hsc70 preserved aberrant Tau, but it remained unknown if selective inhibition of the activity of this Hsp70 isoform could facilitate Tau clearance. Using single point mutations in the nucleotide binding domain, we assessed the effect of several mutations on the functions of human Hsc70. Biochemical characterization revealed that one mutation abolished both Hsc70 ATPase and refolding activities. This variant resembled the ADP-bound conformer at all times yet remained able to interact with cofactors, nucleotides, and substrates appropriately, resembling a dominant negative Hsc70 (DN-Hsc70). We then assessed the effects of this DN-Hsc70 on its client Tau. DN-Hsc70 potently facilitated Tau clearance via the proteasome in cells and brain tissue, in contrast to wild type Hsc70 that stabilized Tau. Thus, DN-Hsc70 mimics the action of small molecule pan Hsp70 inhibitors with regard to Tau metabolism. This shift in Hsc70 function by a single point mutation was the result of a change in the chaperome associated with Hsc70 such that DN-Hsc70 associated more with Hsp90 and DnaJ proteins, whereas wild type Hsc70 was more associated with other Hsp70 isoforms. Thus, isoform-selective targeting of Hsc70 could be a viable therapeutic strategy for tauopathies and possibly lead to new insights in chaperone complex biology.

Keywords: 70-kilodalton heat shock protein (Hsp70); Alzheimer disease; Tau protein (Tau); chaperone; heat shock protein (HSP); inhibitor.

FIGURE 1.

Point mutations in Hsc70 mimicking small molecule inhibition variously affect Tau levels. A, locations of point mutations the ribbon structure (PDB code 3FZF) of the ATP-bound Hsc70 NBD. E175S is blue; all other mutations are shown in green. B, in vitro ATPase activity of human Hsc70 NBD (amino acids 1–386) in the presence of DnaJA2. WT is shown in cyan, F68L, D152K, S208A, C267S are all in gray), and E175S is indicated in blue. C, screen of in vitro DnaJA2-stimulated luciferase refolding activity of human Hsc70 NBD (amino acids 1–386) of WT Hsc70 (cyan) compared with F68L, D152K, S208A, C267S (all gray) and E175S(blue) Hsc70. AU, absorbance units. D, representative Western blot HEK293T cells overexpressing Tau and each full-length Hsc70 variant compared with a mock control. Quantification of Tau levels are shown in E (mean ± S.D., n = 3), *, p ≤ 0.05 by one-way ANOVA. F, overexpression of DN Hsc70 does not significantly alter the number of viable cells at 24 or 48 h. Data are the mean ± S.E., n = 3 independent repeats.

FIGURE 2.

E175S Hsc70 is a dominant negative Hsc70. A, E175S Hsc70 (blue) lacks appreciable ATPase activity compared with WT Hsc70 (cyan) in reactions stimulated with DnaJA1, DnaJB1, or DnaJB4. Data are the means of three independent measurements. B, fluorescence polarization competition assays indicate both ATP and ADP outcompete ATP-FAM binding in E175S Hsc70, indicating the protein binds nucleotide. Data are the means of triplicate independent reactions. C, E175S Hsc70 (blue) does not differ in ability to bind BAG1 compared with WT Hsc70 (cyan) as measured by a flow cytometry protein interaction assay. D, E175S Hsc70 (blue) does not differ from WT Hsc70 (cyan) in BAG1-stimulated fluorescence polarization tracer (ATP-FAM) release competition experiments. Data are the means of three independent measurements. E, E175S (blue) cannot refold luciferase irrespective of DnaJ used to stimulate the reaction when compared with Hsc70 WT (cyan). F, Hsp72 WT or Hsc70 E175S with ATP and ADP or without nucleotide. The addition of ATP reduces substrate HLA-FAM tracer binding in Hsp72 but not Hsc70 E175S. Data represent the means.

FIGURE 3.

Chemical shifts indicate structural rearrangement in E175S Hsc70 nucleotide binding domain. A, overlay of 700 MHz ¹H,¹⁵N TROSY spectra of Hsc70(1–386) in the ATP state (red) and Hsc70(1–386) in the ADP state (blue) on 900 MHz ¹H,¹⁵N TROSY spectra of Hsc70(1–386)E175S in the ATP state (orange) and Hsc70(1–386)E175S in the ADP state (cyan). The spectra show major differences between the mutant and wild type and no differences between the mutant Hsc70 ATP- and ADP-bound state but significant differences between WT Hsc70 ATP- and ADP-bound state. B, enlargement of A, The ¹H,¹⁵N cross-peak for His-227, a residue close to the nucleotide binding site, shifts in the wild type protein between the ATP and ADP state, whereas it does not for Hsc70(1–386)E175S. C, 900-MHz ¹H,¹⁵N TROSY spectra of Hsc70(1–386)E175S in the ATP state (orange) and Hsc70(1–386)E175S in the APO state (green). D, overlay of 900 MHz ¹H,¹⁵N TROSY spectra of Hsc70(1–386)E175S in the ATP state (orange) andHsc70(1–386)E175S in the ADP state (cyan). E, overlay of 700 MHz ¹H,¹⁵N TROSY spectra of Hsc70(1–386) in the ATP state (red) and Hsc70(1–386) in the ADP state (blue). F, overlay of 700 MHz ¹H,¹⁵N TROSY spectra of Hsc70(1–386) in the ADP state (red) and Hsc70(1–386) in the APO state (green). G, NH chemical shift differences between WT-Hsc70 NBD and Glu-175 Hsc70 NBD, both in the ADP-bound state, plotted on the crystal structure of Hsc70 NBD (PDB code 3HSC). Changes in structure mapped on the ribbon structure of Hsc70 NBD reveal E175S induced structural rearrangement in subdomains IA and IIA. Green, no change; yellow, shift; red, loss of resonances; gray, no information. Purple indicates the location of E175S with ADP and phosphate (blue) and a Mg²⁺ ion (orange).

FIGURE 4.

E175S does not undergo interdomain allosteric conformational changes. ¹H-detected ¹⁵N-R₁ relaxation for WT Hsc70 (900 MHz) is shown. The relaxation times were 100, 300, 600, 1000, 2000, and 3000 ms (labeled at peak heights). Tabulated results ± uncertainty values (estimated by jackknife procedure (41)) for full-length WT Hsc70 and E175S Hsc70 are shown below the spectra.

FIGURE 5.

Dominant negative Hsc70 reduces Tau levels. A, Western blot of the effects of shRNA-mediated knockdown of Hsc70 on Tau levels in HEK293T cells. B, confocal microscopy of HEK293T cells overexpressing RFP-Tau (red) in the presence of vector, WT Hsc70-FLAG, or E175S Hsc70-FLAG (green). Confocal z-stack images at 60×; scale bar is 10 μm. Quantification of RFP-Tau intensity levels ± S.E. are shown. *, p < 0.05, ** p < 0.01, one way ANOVA with Tukey's post hoc test for multiple comparisons. C, Western blot of the effects of overexpression of WT and DN Hsc70-FLAG on Tau levels in HEK293T cells. Quantification of Tau levels ± S.E. is shown. *, p < 0.05, one way ANOVA. D, MAP2 levels in cells overexpressing WT Hsc70 and E175S Hsc70. Quantification of MAP2 levels ± S.E. are shown.

FIGURE 6.

DN Hsc70 facilitates Tau degradation in neurons. Tau levels in organotypic hippocampal slices from FVB-Tg(tetO-MAPT*P301L) mice transduced with either AAV9-GFP, WT Hsc70-FLAG, or E175S Hsc70-FLAG and immunostained with anti-FLAG (green), anti-Tau H150 (red), and DAPI (blue). The scale bar is 10 μm (60× objective with 2× zoom). Data are the mean intensity ± S.E.; n = 10; ***, p < 0.001, one-way ANOVA.

FIGURE 7.

DN Hsc70 degrades Tau via the proteasome. A, Western blot image of Tau immunoprecipitated (IP) from cells expressing vector, WT Hsc70, or E175S. Shown is quantification of Hsp90 levels in the Tau immunoprecipitation; data are the mean ± S.E. B, Western blot of Tau levels in HEK293T cells transfected with WT Tau and a transfection control, Hsc70 WT, or Hsc70 E175S after inhibition of the proteasome by 6 h of epoxomicin treatment. Quantification data are the mean ± S.E., n = 4, *, p < 0.05. C, Western blot of inhibition of macroautophagy by Beclin1 knockdown; at least 60% knockdown was achieved with the siRNA. Quantification is the mean ± S.E., n = 3.

FIGURE 8.

Mass spec reveals a changing chaperome for DN Hsc70 compared with WT Hsc70. A, average spectral counts plotted as percentages to illustrate whether a protein was enriched in WT (cyan) or E175S Hsc70 (blue). Data are the averages of three independent mass spectroscopy experiments. Results that were <10 spectral counts in either sample were discarded to ensure robustness of the results. B, confirmation of mass spectroscopy results; representative Western blot images of Tau immunoprecipitated (IP) from cells overexpressing Hsc70 WT Hsc70 or E175S Hsc70 and probed for DnaJA1 and DnaJC7 is shown.