Chaperone-mediated 26S proteasome remodeling facilitates free K63 ubiquitin chain production and aggresome clearance

Abstract

Efficient elimination of misfolded proteins by the proteasome system is critical for proteostasis. Inadequate proteasome capacity can lead to aberrant aggregation of misfolded proteins and inclusion body formation, a hallmark of neurodegenerative disease. The proteasome system cannot degrade aggregated proteins; however, it stimulates autophagy-dependent aggregate clearance by producing unanchored lysine (K)63-linked ubiquitin chains via the proteasomal deubiquitinating enzyme Poh1. The canonical function of Poh1, which removes ubiquitin chains en bloc from proteasomal substrates prior to their degradation, requires intact 26S proteasomes. Here we present evidence that during aggresome clearance, 20S proteasomes dissociate from protein aggregates, while Poh1 and selective subunits of 19S proteasomes are retained. The dissociation of 20S proteasome components requires the molecular chaperone Hsp90. Hsp90 inhibition suppresses 26S proteasome remodeling, unanchored ubiquitin chain production, and aggresome clearance. Our results suggest that 26S proteasomes undergo active remodeling to generate a Poh1-dependent K63-deubiquitinating enzyme to facilitate protein aggregate clearance.

Keywords: aggresome; autophagy; deubiquitylation (deubiquitination); histone deacetylase 6 (HDAC6); proteasome; ubiquitin.

FIGURE 1.

20S proteasomes dissociate from the aggresome during de-aggregation. A, immunolocalization of 19S-associated deubiquitinase Poh1 (red) and (B) 20S-associated PSMA2 (red), both co-stained with ubiquitin (green) as the aggresome marker. Proteasome subunits were observed upon aggresome formation at 24 h of MG132 treatment and 12 h of MG132 washout when aggresomes undergo de-aggregation. Boxed regions of the image are zoomed to highlight proteasome associations with the aggresome. Scale bar indicates 15 μm. C, immunostaining results from A and B were quantified at 24 h MG132 and 12 h wash for both subunits. Refer to “Experimental Procedures” for image analysis. Significant loss of PSMA2 is observed during 12 h wash, whereas Poh1 signal remains stable. Error bars indicate ± S.E. *, p < 0.01. D, immunoblotting of 19S DUB (Poh1) and 20S core subunits (PSMA2, PSMA4, PSMB4, and PSMB5) separated by detergent-soluble and insoluble (aggresome) fractions. Actin is provided as a loading control and detergent resistant Hsp70 and Hsp90 reflect level of insoluble aggresome. Indicated densitometry values for each protein reflect the density ratio: (band density for a particular treatment condition/band density at 24 h MG132). All ratios were normalized to actin. E, densitometry data displayed in D were quantified for the insoluble fraction at 24 h MG132 and 12 h wash averaged over three separate experiments. Error bars indicate ± S.E. *, p < 0.01.

FIGURE 2.

19S ATPase dissociate while non-ATPase subunits remain with de-aggregated aggresomes. A, immunolocalization of 19S-associated ATPase RPT3 (red), and (B) non-ATPase RPN10 (red), at 24 h MG132 and 12 h Wash. Both subunits were co-stained with either ubiquitin or p62 (green) as the aggresome marker, respectively. Note that RPN10 displays weak permeabilization of the aggresome upon formation at 24 h MG132, but staining improves upon 12 h MG132 wash off, which may indicate ease of antibody accessibility upon proteasome remodeling. ATPase subunit RPT3 displays clear dissociation from de-aggregated aggresomes. Boxed regions of the images are zoomed to highlight proteasome associations with the aggresome. Scale bar indicates 15 μm. C, immunoblotting of 19S lid (Poh1, RPN7, RPN8, and RPN9), 19S base non-ATPase (RPN1, RPN2, and RPN10) and ATPase (RPT6 and RPT3) subunits separated by detergent-soluble and insoluble (aggresome) fractions. These bands were developed on the same gel as Fig. 1 to clearly allow comparison of relative abundance of 19S and 20S subunits during formation and clearance. Indicated densitometry values reflect ratio of each band density to the density of 24h MG132 condition and normalized to actin. D, densitometry data displayed in C were quantified for the insoluble fraction and averaged over three separate experiments. Error bars indicate ± S.E. *, p < 0.01.

FIGURE 3.

20S proteasome is not essential for aggresome clearance. A, aggresomes labeled with ubiquitin (red) 24 h post MG132 wash with or without the addition of nocodazole under non-targeting siRNA control (Control KD), Poh1 KD, RPN10 KD, PSMA2 KD, or PSMB4 KD. Nocodazole-sensitive PSMA2 KD aggregates reflect aggregates undergoing aggresome formation. Knockdown efficiency is displayed by immunoblotting adjacent to each knockdown condition. B, quantification of aggresomes and dispersed aggregates displayed in A over three different experiments. Error bars indicate ± S.E. *, p < 0.01. C, HDAC6 associates with micro-aggregates in PSMA2 KD cells under 24 h wash with nocodazole, which classifies those dispersed aggregates as pre-aggresomal. Scale bar for all image data indicates 15 μm.

FIGURE 4.

Hsp90 facilitates 26S proteasome remodeling at the aggresome. A, immunolocalization of Hsp90 at the aggresome upon formation. B, immunostaining of 19S (Poh1) and (C) 20S (PSMA2) upon aggresome formation (24 h MG132) and 12 h of MG132 washout with Hsp90 inhibitor 17-DMAG (2 μm). Aggresomes are marked with ubiquitin (green). PSMA2 does not display a significant dissociation from aggresomes in the presence of 17-DMAG during MG132 washout. Boxed regions of the image are zoomed to highlight proteasome associations with the aggresome. These experiments were conducted at the same time as Fig. 1, A and B to minimize variations in staining between experiments. Scale bar indicates 15 μm. D, quantification of signal intensity for data displayed in B and C. Refer to “Experimental Procedures” for image analysis. No significant reduction in PSMA2 was observed under 17-DMAG. E, immunoblotting of 19S (Poh1, RPN7, RPN8, RPN9, RPN10, RPN1, RPN2, RPT3, and RPT6) and 20S (PSMA2, PSMA4, PSMB4, and PSMB5) subunits separated by detergent soluble and insoluble fractions. Note that compared with 12 h wash, 20S and ATPase subunits do not dissociate in the presence of 17-DMAG. Actin is provided as a loading control and detergent-resistant Hsp70 and Hsp90 reflect the level of insoluble aggresome. Hsp70 expression has been previously reported to increase under Hsp90 inhibition. Densitometry values reflect ratios of band density for each condition to the intensity of 24 h MG132 treatment. All ratios are normalized to actin. F, densitometry data for results displayed in E quantified over three separate experiments. Error bars indicate ± S.E. *, p < 0.01.

FIGURE 5.

Hsp90 is required for efficient aggresome clearance. A, Hsp90 inhibition by 17-DMAG (2 μm), Novobicin (500 μm), 17-AAG (2 μm), PU-H71 (2 μm), HS-10 (1 μm) prevents aggresome clearance 24 h post MG132 washout. Aggresomes are labeled with ubiquitin (red). B, presence of aggresomes was quantified among three separate experiments and compared with DMSO as a control. Error bars indicate ± S.E. *, p < 0.01. C, aggresomes remain intact even in the presence of nocodazole during 24 h wash with 17-DMAG, which reflects a clear defect in aggresome clearance. D, aggresomes remaining under nocodazole wash with or without 17-DMAG are quantified among three separate experiments. Error bars indicate ± S.E. *, p < 0.01. Scale bars indicates 15 μm.

FIGURE 6.

HDAC6 inhibition does not affect dissociation of 20S proteasomes. A, immunostaining of 19S (Poh1) and 20S (PSMA2) upon aggresome formation (24 h MG132) and 12 h of washout with HDAC6 inhibitor Tubastatin-A (10 μm) (TBSA). PSMA2 displays significant dissociation from aggresomes in the presence of TBSA during MG132 washout. Boxed regions of the image are zoomed to highlight proteasome associations with the aggresome. These experiments were done at the same time as Fig. 1, A and B to minimize variations in staining between experiments. B, image data from A are quantified among three separate experiments. Refer to “Experimental Procedures” for image analysis. Error bars indicate ± S.E. *, p < 0.01. Scale bars indicates 15 μm. C, immunoblotting of Poh1 and PSMA2 in the detergent-soluble and insoluble fractions at 24 h MG132, 12 h wash, and 12 h wash with TBSA. Addition of TBSA does not affect the dissociation of PSMA2 from the aggresome. Densitometry readings reflect ratio of band density for each condition to 24 h MG132. All ratios are normalized to actin.

FIGURE 7.

Hsp90 facilitates the efficient production of free K63 ubiquitin chains and HDAC6 activation. A, immunoblotting of K63 linked ubiquitin in the detergent-soluble and insoluble fractions during conditions of DMSO, 24 h MG132, 12 h wash, and 12 h wash with 17-DMAG. There is a clear retention of K63 ubiquitin on the aggresome under inhibition of Hsp90. B, densitometry quantification of data displayed in A calculated by the ratio of band density for each condition to band density at 24 h MG132. Data quantified over three separate experiments, error bars indicate ± S.E. *, p < 0.01. C, FLAG-HDAC6 was immunoprecipitated under the following conditions: 1) no treatment, 2) 5 μm MG132 for 24h, 3) 12h MG132 washout, and 4) 12 h MG132 washout in the presence of 2 μm 17-DMAG. The immune complexes were subjected to heating, and the eluates were treated with isopeptidase T (IsoT) as indicated (see the “Experimental Procedures” for details). Samples were analyzed by immunoblotting with antibody for ubiquitin. Note that MG132 washout (condition 2 versus 3) led to a decrease in total ubiquitinated protein levels in whole cell lysate (input) but an increase in HDAC6-associated free ubiquitin chains. This increase was suppressed under the addition of 17-DMAG (condition 4). D, densitometry quantification of data displayed in C. There is a significant reduction in the amount of free ubiquitin chains bound by HDAC6 in the presence of 17-DMAG. Data reflect the ratio of density of each condition to the density at 24 h MG132 treatment across three separate experiments. Error bars indicate ± S.E. *, p < 0.01. E, immunolocalization of HDAC6 upon aggresome formation (24 h MG132), de-aggregation (12 h Wash), and washout under Hsp90 inhibition (12 h 17-DMAG). Note that HDAC6 dissociation is blocked under 17-DMAG reflecting a state of inactivation. Boxed regions were zoomed and displayed below each condition. Scale bar indicates 15 μm. F, imaging data from E is quantified among three separate experiments. Error bars indicate ± S.E. *, p < 0.01. Refer to “Experimental Procedures” for image analysis.