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. 2013 Feb;9(2):112-8.
doi: 10.1038/nchembio.1140. Epub 2012 Dec 9.

Activation of Hsp70 Reduces Neurotoxicity by Promoting Polyglutamine Protein Degradation

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Free PMC article

Activation of Hsp70 Reduces Neurotoxicity by Promoting Polyglutamine Protein Degradation

Adrienne M Wang et al. Nat Chem Biol. .
Free PMC article

Abstract

We sought new strategies to reduce amounts of the polyglutamine androgen receptor (polyQ AR) and achieve benefits in models of spinobulbar muscular atrophy, a protein aggregation neurodegenerative disorder. Proteostasis of the polyQ AR is controlled by the heat shock protein 90 (Hsp90)- and Hsp70-based chaperone machinery, but mechanisms regulating the protein's turnover are incompletely understood. We demonstrate that overexpression of Hsp70 interacting protein (Hip), a co-chaperone that enhances binding of Hsp70 to its substrates, promotes client protein ubiquitination and polyQ AR clearance. Furthermore, we identify a small molecule that acts similarly to Hip by allosterically promoting Hsp70 binding to unfolded substrates. Like Hip, this synthetic co-chaperone enhances client protein ubiquitination and polyQ AR degradation. Both genetic and pharmacologic approaches targeting Hsp70 alleviate toxicity in a Drosophila model of spinobulbar muscular atrophy. These findings highlight the therapeutic potential of allosteric regulators of Hsp70 and provide new insights into the role of the chaperone machinery in protein quality control.

Conflict of interest statement

Competing Financial Interest Statement:

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Hip increases client protein ubiquitination and promotes AR112Q clearance
(a) Hip promotes nNOS ubiquitination. HEK293T cells transiently expressing nNOS, HA-ubiquitin (HA-Ub) and increasing amounts of Hip were treated with lactacystin (24 hr). nNOS was immunoprecipitated from cytosolic lysates, and Ub-nNOS from three separate experiments was quantified (mean ± SEM). ***P<0.001. (b, c) Hip decreases AR112Q levels. (b) HeLa cells transiently expressing AR112Q and increasing amounts of Hip were treated with R1881 (10 nM) for 24 hr. Lysates were separated into supernatant and 15,000 g pellet fractions, then analyzed by western blot (left) (For uncropped gel images, see Supplementary Figure 12). At right, pelleted AR from three experiments was quantified. n.s. = not significant.(c) HeLa cells were cotransfected with AR112Q and Hip, then treated with R1881 (10 nM) for 24 hrs prior to 8 hr treatment with MG132 (10 μM). (d) Hip diminishes AR112Q aggregates. PC12 cells expressing tet- regulated AR112Q were transfected with FLAG-Hip, then treated with doxycycline and R1881 (10 nM) for 24 hr. AR and FLAG-Hip were visualized by confocal microscopy (left). Scale bar, 10 μm. The expression of Hip significantly decreased the frequency of polyQ AR intranuclear inclusions (right, mean ± SEM).
Figure 2
Figure 2. YM-1 increases Hsp70 binding to a denatured substrate and competes with Hip for binding
(a) Chemical structures of YM-1 and the two biotinylated probes, 2 and 3. (b) YM-1 promotes binding of Hsp70 to denatured luciferase. Binding of purified Hsp70 to denatured luciferase was measured using an HRP-coupled, anti-Hsp70 antibody. A solvent control is shown for comparison. Data are mean± SEM.(c) Partial trypsin proteolysis shows that YM-1 favors an ADP-like conformation. Full length human Hsp70 was treated with ADP, ATP or ATP and a J domain (Jd). While proteolysis of the ADP-treated sample produces a single characteristic band (band 2), addition of ATP yields an additional fragment (band 1). The ratio of bands 1 and 2 are quantified and the error of duplicate experiments is shown. *P<0.005.(d) Binding of the biotinylated probes to Hsp70 confirms that the pyridine ring of YM-1 is solvent exposed. Experiments were carried out in triplicate. Data are mean ± SEM. (e) Binding of 2 to Hsp70 is specific and can be competed with unlabelled YM-1. Experiments were carried out in triplicate. Data are mean ± SEM.(f) YM-1 and Hip bind competitively to Hsp70. Binding of 2 to immobilized Hsp70 is diminished by pre-incubation with increasing amounts of Hip. Experiments were carried out in triplicate. Data are mean ± SEM.
Figure 3
Figure 3. YM-1 increases client protein ubiquitination and diminishes AR112Q aggregation
(a) YM-1 promotes nNOS ubiquitination. HEK293 cells stably expressing nNOS were treated with increasing amounts of YM-1 and lactacystin for 24 hours. Short exposure of input (on left) shows unmodified nNOS, while longer exposure shows an accumulation of a higher molecular weight (MW) species representing mono-ubiquitinated nNOS (highlighted in rectangle). (For uncropped gel images, see Supplementary Figure 12) Quantification of mono-ubiquitinated nNOS from four separate experiments is shown in the middle, **P<0.01, ***P<0.001. Immunoprecipitation (on right) shows increased nNOS poly-ubiquitination in the presence of YM-1. (b) YM-1 decreases insoluble AR112Q. PC12 cells expressing tet-regulated AR112 were treated with R1881 (10 nM) and YM-1 for 16 hours. Pelleted (15,000 g) AR112Q was visualized by immunoblot analysis and data from three separate experiments was quantified (mean ± SEM). *P<0.05, **P<0.01(c) PC12 cells were treated as in (b). AR was visualized by fluorescence microscopy and cells with nuclear inclusions quantified. Data are mean ± SEM. **P<0.01.
Figure 4
Figure 4. YM-1 increases Hsp70-dependent degradation of AR112Q
(a) PC12 cells were induced to express AR112Q in the presence of R1881 (10 nM) for 48 hr, washed to remove doxycycline to turn off the transgene, and then incubated in the presence or absence of YM-1. Cells were stained for AR (left). Scale bar, 10 μm. Quantification of field signal intensity from three experiments (right). Data are mean ± SEM. ***P<0.001 (b) YM-1 promotes clearance of insoluble and oligomeric AR112Q. Signal intensity of AR112Q monomer in the 15,000 g pellet and high MW oligomers in the soluble fraction after ultracentrifugation was quantified from triplicate experiments (mean ± SEM). *P<0.05 (c) Immunoblot of AR112Q in supernatant and pellet shows that effects of YM-1 are blocked by 24 hr treatment with MG132 (10 μM). (For uncropped gel images, see Supplementary Figure 12.)(d) HeLa cells treated with vehicle, YM-1 or geldanamycin (GA) for 24 hr were probed for expression of inducible Hsp70, Hsp40 and Hsp25. (e) Levels of Akt and ERK1/2 are unchanged and glucocorticoid receptor (GR) mildly decreased by treatment of PC12 cells with YM-1, using the lysates probed in panel b.(f) YM-1 effects are dependent upon Hsp70. PC12 cells expressing tet-regulated AR112Q were transfected with siRNAs targeted at inducible Hsp70 or nontargeted control. After 24 hrs, cells were treated with R1881 (10 nM) plus YM-1 or vehicle. Effects of YM-1 on pelleted AR112Q are shown on left and quantified from triplicate experiments on right. Data are mean ± SEM. *P<0.05.
Figure 5
Figure 5. Hsp70 allosteric activators rescue toxicity in Drosophila expressing AR52Q
(a-c) Representative scanning electron micrographs (SEMs) of eyes from GMR-Gal4;UAS-hAR52Q flies reared at 29° C. Lower panels show high magnification images from posterior aspects of eyes. Scale bars: upper panels, 100 μm; lower panels 10 μm. (a) Flies reared on food supplemented with vehicle control. (b) Flies reared on food containing dihydrotestosterone (1 mM DHT). (c) Flies reared on food containing DHT plus YM-1 (1 mM). (d) SEMs were scored for the presence of more than 1 bristle at inter-ommatidial junctions (mean ± SEM, n = 150-200 junctions/condition). *P<0.05, **P<0.01. (e) DHT-dependent toxicity is rescued by YM-1. Reported are the percent of pupae that fail to eclose (mean ± SEM, n = 200-800/condition) when reared on DHT or vehicle. Flies compared are BG380-Gal4;;WCS (white bars), BG380-Gal4;;UAS-hAR52Q (black bars), and BG380-Gal4;;UAS-hAR52Q reared on YM-1 (gray bars). **P<0.01, ***P<0.001. (f) Overexpression of HIP-R suppresses toxicity. Reported are the percent of pupae that fail to eclose (mean ± SEM, n = 200-800/condition) when reared on DHT or vehicle. Flies compared are OK371-Gal4;WCS (white bars), HIP- R;OK371-Gal4 (striped bars), OK371-Gal4;UAS-hAR52Q (black bars), and HIP- R;OK371-Gal4;UAS-hAR52Q (gray bars). ***P<0.001.
Figure 6
Figure 6. Model of the Hsp90/Hsp70-based chaperone machinery and regulation of polyQ AR degradation
Hsp90 and Hsp70 form a heterocomplex to stabilize the polyQ AR, enable ligand binding (depicted as white steroid within AR) and guide intracellular localization (top left). Dissociation of Hsp90 following the addition of small-molecule inhibitors or ligand-dependent conformation change of the polyQ AR permits unfolding of the mutant protein. Substrate-bound Hsp70 then recruits chaperone dependent ubiquitin ligases such as CHIP to promote degradation through the proteasome. We note that CHIP and Hip both bind Hsp70 via tetratricopeptide repeat domains, although it is unknown whether this binding occurs simultaneously. Furthermore, we note that other chaperone dependent ubiquitin ligases may function redundantly with CHIP. We demonstrate here that allosteric activators of Hsp70, including Hip and YM-1 (in green), increase substrate binding affinity, facilitate client protein ubiquitination and promote polyQ AR clearance by the proteasome. This strategy alleviates polyglutamine toxicity by facilitating degradation of the mutant protein. The broken line for Hsp70 in the final heterocomplex indicates that it is present in substoichiometric levels with respect to the receptor. IMM, immunophilin.

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