A cellular system that degrades misfolded proteins and protects against neurodegeneration

Abstract

Misfolded proteins compromise cellular function and cause disease. How these proteins are detected and degraded is not well understood. Here we show that PML/TRIM19 and the SUMO-dependent ubiquitin ligase RNF4 act together to promote the degradation of misfolded proteins in the mammalian cell nucleus. PML selectively interacts with misfolded proteins through distinct substrate recognition sites and conjugates these proteins with the small ubiquitin-like modifiers (SUMOs) through its SUMO ligase activity. SUMOylated misfolded proteins are then recognized and ubiquitinated by RNF4 and are subsequently targeted for proteasomal degradation. We further show that PML deficiency exacerbates polyglutamine (polyQ) disease in a mouse model of spinocerebellar ataxia 1 (SCA1). These findings reveal a mammalian system that removes misfolded proteins through sequential SUMOylation and ubiquitination and define its role in protection against protein-misfolding diseases.

Figure 1. PML promotes the degradation of Atxn1 82Q and other nuclear misfolded proteins

(A) HeLa cells transfected with Atxn1 82Q-GFP were stained with anti-PML antibody (red) and DAPI (blue). Individual and merged images are shown. Scale bar: 10 μm. (B) Atxn1 82Q-GFP was expressed alone or together with PML in HeLa cells. Left: representative fluorescence images of cells. Scale bar: 20 μm. Right: quantification of cells based on sizes of Atxn1 82Q-GFP inclusions. (C) Atxn1 82Q-GFP was expressed alone or together with PML in HeLa cells (left), or alone in HeLa cells that were previously treated with control (-) or PML siRNA. Cell lysate fractions (when indicated) and whole cell lysates (WCL) were analyzed by filter retardation assay (for SR fraction) or Western blot (WB; for the rest). Molecular weight standards (in kDa) and relative ratios of SS or SR Atxn1 versus actin are indicated. (D) Steady-state levels of FLAG-Atxn1 82Q or 30Q when expressed alone or together with PML in HeLa cells (left), or when expressed alone in HeLa cells that were treated with control or PML siRNA (right), analyzed by WB. (E) Effect of PML on the stability of total FLAG-Atxn1 82Q protein, analyzed by a pulse chase assay and autoradiography. The relative amounts of ³⁵S-labeled Atxn1 82Q is indicated. (F and G) Effect of PML overexpression (F) and knockdown (G) on the stability of Atxn1 82Q-GFP, analyzed by CHX treatment and WB. (H) Effect of PML on Atxn1 82Q-GFP levels in the absence or presence of MG132. (I) Top: Relative percentages of Httex1p 97QP-expressing cells with cytoplasmic (left) and nuclear (right) inclusions, in the absence or presence of PML (means + SD, n = 3). Bottom: Representative fluorescence images of transfected cells immunostained with an anti-Htt antibody. Arrowheads indicate Httex1p 97QP aggregates. (J and K) Levels of HA-Httex1p 97QP and HA-Httex1p 97QP(KR) (J) and GFP-TDP-43 (K) in cells with and without PML over-expression. Virtually Htt aggregates were in the SR fraction. (L) Stability of nFucDM-GFP in control and PML-depleted cells, analyzed by CHX treatment and WB. See also Figure S1.

Figure 2. Recognition of misfolded proteins by PML

(A) Binding of GST-Htt 25Q and GST-Htt 103Q to immobilized FLAG-PML and FLAG-GFP (negative control), analyzed by an in vitro pull down assay followed by WB (top and bottom) and Ponceau S staining (middle). *, IgG heavy chain. (B) Binding of GST-PML (shown on the right) and the control GST protein to native (N) and urea-denatured (D) luciferase (luc) immobilized on Ni-NTA beads, analyzed as in (A). *, Non-specific proteins from BL21 bacterial lysates that bound to the control beads. (C) Binding of indicated GST-Htt fusions to FLAG-PML CC conjugated on anti-Flag M2 beads or to control beads, analyzed by WB (top and middle) and Coomassie staining (bottom). (D) Binding of PML F12/SRS2 (shown on the right) to peptide library derived from luciferase. The N-terminal amino acid of the first peptide and the number of the last peptide spotted in each row are indicated. (E) The occurrence of each amino acid in PML SRS2-binding peptides relative to its occurrence in the luciferase peptide array (set at 100%). See also Figures S2 and S3.

Figure 3. SUMO2/3 is involved in the ubiquitination and PML-mediated degradation of Atxn1 82Q

(A) SUMO1- and SUMO2/3-modification of Atxn1 82Q by in HeLa cells treated without or with MG132. For better comparison of modified Atxn1 82Q, IP products with a similar level of unmodified Atxn1 82Q were analyzed by WB. *: non-specific bands. (B) Localization of Atxn1 82Q (detected by anti-FLAG antibody, red) in GFP-SUMO2- or GFP-SUMO3-expressing U2OS cells treated with or without MG132. Scale bar: 10 μm. (C) SUMO2/3 modification of Atxn1 82Q and 30Q in HeLa cells, analyzed by d-IP followed by WB (top) and Ponceau S staining (bottom). (D) Atxn1 82Q was expressed alone or together with SUMO1 or HA-SUMO2 KR in HeLa cells that were previously transfected with the indicated siRNA or un-transfected (-). Cells were treated with or without MG132. SUMOylation and ubiquitination of FLAG-Atxn1 82Q was analyzed by d-IP and WB. (E) Levels of Atxn1 82Q-GFP expressed alone or together with increasing amounts of PML in HeLa cells pre-treated with the indicated siRNA. (F) Effect of PML on Atxn1 82Q-GFP levels in the presence or absence of SUMO1 or SUMO2 KR. See also Figure S4.

Figure 4. PML promotes SUMOylation of Atxn1 82Q

(A) SUMOylation of FLAG-Atxn1 82Q in HeLa cells in the absence or presence of HA-PML cells, and without or with MG132 treatment. The amount of Atxn1 82Q DNA used for transfected were adjusted to yield comparable levels of the unmodified protein. (B and C) SUMOylation of FLAG-Atxn1 82Q (B) and FLAG-nFlucDM-GFP (C) in control and PML siRNA-transfected HeLa cells treated without or with MG132. (D and E) SUMOylation of purified HA-Atxn1 82Q-FLAG was performed in the presence of recombinant FLAG-PML, FLAG-PML M6, and SUMO2 as indicated. In (D), the amounts of d-IP products were adjusted to yield a similar level of unmodified Atxn1 82Q (middle panel). (F) Levels of Atxn1 82Q-GFP in HeLa cells in the absence or presence of increasing amounts of PML or PML M6. See also Figure S4.

Figure 5. A role of RNF4 in the degradation of Atxn1 82Q

(A) Levels of Atxn1 82Q-GFP in HeLa cells without and with RNF4 overexpression. (B and C) Effect of RNF4 overexpression on Atxn1 82Q-GFP stability in control and PML-depleted HeLa cells, analyzed by CHX treatment and WB. Relative SS Atxn1 82Q/actin ratios are shown in (C). (D) Levels of FLAG-Atxn1 82Q in HeLa cells pretreated with a control siRNA (-) or a combination of three RNF4 siRNAs (D). (E) Representative fluorescent images of Atxn1 82Q-GFP in control and RNF4-knock down HeLa cells. Scale bar: 20 μm (F) Localization of Atxn1 82Q-GFP and endogenous RNF4 in HeLa cells treated with vehicle (DMSO) or MG132. Scale bar: 10 μm. (G and H) Levels of FLAG-Atxn1 82Q and FLAG-Atxn1 30Q (G) or HA-Httex1p 97QP and HA-Httex1p 97QP(KR) (H) in HeLa cells without and with RNF4 overexpression. (I) Stability of nFlucDM-GFP in HeLa cells stably expressing shCtrl and shRNF4 (left) and the levels of RNF4 in these cells (right). See also Figure S5.

Figure 6. RNF4 promotes the ubiquitination and degradation of SUMO2/3-modified Atxn1 82Q

(A and B) Levels of SUMOylated FLAG-Atxn1 82Q (A) and FLAG-nFlucDM-GFP (B), in the absence or presence of RNF4, in HeLa cells treated with or without MG132. d-IP products with similar levels of unmodified proteins and WCL were analyzed. (C) Levels of SUMOylated FLAG-Atxn1 82Q in HeLa cells that were pre-treated with a control siRNA or a combination of RNF4 siRNAs, analyzed as in (A). (D and E) Unmodified and SUMO2-modified FLAG-Atxn1 82Q proteins conjugated on M2 beads (+), or control M2 beads (-), were incubated with ubiquitination reaction mixture, in the absence or presence of GST-RNF4. (D) A schematic diagram of the experimental design. (E) WB analysis of FLAG-Atxn1 82Q (left) and GST-RNF4 (right). (F and G) Localization of Atxn1 82Q-GFP and RNF4 proteins (detected by anti-FLAG antibody) in HeLa. Scale bar: 10 μm. (H) Effect of the indicated RNF4 proteins on Atxn1 82Q-GFP levels in HeLa cells. (I) Effect of PML overexpression on Atxn1 82Q-GFP levels in HeLa cells that were pre-treated with control or RNF4 siRNA. See also Figure S6.

Figure 7. PML deficiency exacerbates behavioral and pathological phenotypes of SCA1 mouse model

(A and B) Retention times (average + standard error of the means or SEM) on accelerating Rotarod at 7 (A) and 11 (B) weeks of age, with the number of animals indicated in parenthesis. (C and D) Cerebellar sections of 12-week-old animals were stained with hematoxylin. (C) Quantification of molecular layer thickness (means + SEM, n = 3 mice/genotype). (D) Representative images of the staining. Scale bar: 200 μm. (E and F) Cerebellar sections of 1-yr-old animals were stained with an anti-calbindin antibody. (E) Quantitation of Purkinje cells, graphed as the average number of soma per 1 mm length (means + SEM, n = 4 mice/genotype). (F) Representative images of the staining. Scale bar: 200 μm. (G and H) Sections of the cerebellar cortex from 12-week-old mice were stained with an anti-ubiquitin antibody and counterstained with hematoxylin. (G) Percentage of Purkinje cells with aggregates (means + SEM, n = 3 mice/genotype). (H) Representative images of the immunohistochemistry staining. Arrowheads indicate the ubiquitin positive aggregates in Purkinje cell bodies. Scale bar: 50 μm. No ubiquitin positive aggregates were observed in Purkinje cells in mice without Atxn1^tg/− (Figure S7D). (I) A model for PQC by the PML-RNF4 system. PML recognizes misfolded proteins and conjugates them with poly-SUMO2/3 chain through its SUMO E3 ligase activity (a). RNF4 ubiquitinates SUMOylated-misfolded proteins (b) and targets them for proteasomal degradation (c). See also Figure S7.