Rheb controls misfolded protein metabolism by inhibiting aggresome formation and autophagy - PubMed (original) (raw)

Rheb controls misfolded protein metabolism by inhibiting aggresome formation and autophagy

Xiaoming Zhou et al. Proc Natl Acad Sci U S A. 2009.

Abstract

Perinuclear aggresome formation is a key mechanism to dispose of misfolded proteins that exceed the degradative capacity of ubiquitin-proteasome and autophagy-lysosome systems. Functional blockade of either degradative system leads to an enhanced aggresome formation. The tuberous sclerosis complex-Ras homologue enriched in brain-mammalian target of rapamycin (TSC-Rheb-mTOR) pathway is known to play a central role in modulating protein synthesis and autophagy. However, in spite of the constitutive activation of mTOR and the abrogated autophagy activity in TSC1- or TSC2-deficient cells, the TSC mutant cells are defective in aggresome formation and undergo apoptosis upon misfolded protein accumulation both in vitro and in vivo. High Rheb activity in TSC mutant cells inhibits aggresome formation and sensitizes cell death in response to misfolded proteins. Surprisingly, this previously unrecognized function of Rheb is independent of TOR complex 1. Active Rheb disrupts the interaction between dynein and misfolded protein cargos, and therefore blocks aggresome formation by inhibiting dynein-dependent transportation of misfolded proteins. This study reveals a function of Rheb in controlling misfolded protein metabolism by modulating aggresome formation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

TSC1−/− MEF is defective in aggresome formation. (A) TSC1−/− cells have less insoluble ubiquitinated protein, even in the presence of rapamycin (Rapa). TSC1−/− MEF was a spontaneously immortalized MEF cell line derived from TSC1−/− embryos. TSC1+/+ and TSC1−/− MEFs with or without 20 nM rapamycin pretreatment for 36 h were treated with DMSO or 5 μM MG132 for 4 or 9 h before harvest. RIPA-insoluble cell lysates were normalized and blotted for ubiquitin (UB). RIPA-soluble lysates were blotted for UB, LC3, and Akt (for loading control). (B) Rapamycin does not restore aggresome formation in TSC1−/− cells. TSC1+/+ and TSC1−/− MEFs with or without 20 nM rapamycin pretreatment for 36 h were treated with DMSO or 5 μM MG132 for 9 h before fixation. Cells were immunostained for ubiquitin–protein conjugates (red), and nuclei were counterstained with DAPI (blue). (C) TSC1−/− cells are defective in autophagy and aggresome formation. TSC1+/+, TSC1−/− MEFs expressing EGFP-LC3 (green) treated with 5 μM MG132 or DMSO for 10 h were immunostained for ubiquitin–protein conjugates. Nuclei were stained with DAPI. The data show that LC3 signals encroach aggresome in TSC1+/+ but not in TSC1−/− cells. O/L denotes overlay.

Fig. 2.

Fig. 2.

Rheb regulates ubiquitinated aggresome formation. (A) Rheb regulates endogenous aggresome formation. YFP-Rheb-Q64L (constitutively active) or YFP-Rheb-D60K (dominant-negative) constructs were transfected into A549 cells, followed by 5 μM MG132 treatment for 12 h. (Left) Cells were immunostained for ubiquitin (UB)–protein conjugates, and nuclei were stained with DAPI. (Right) Percentage of aggresome-harboring cells was counted among the transfectants and nontransfectants. Values represent means ± SD of 3 independent experiments. **, P < 0.01 (Student's t test). (B) Rheb regulates aggregation of CFTR-ΔF508. At 18 h after cotransfection of EGFP-CFTRΔF508 with myc-Rheb-Q64L, myc-Rheb-D60K, or control vector, COS-7 cells were immunostained for MYC and nuclei (DAPI). (Left) Representative images are shown from 3 independent experiments. (Right) Percentage of aggresome-harboring cells was scored among the cotransfectants. Values represent means ± SD of 3 independent experiments. *, P < 0.05. (C) Rheb does not regulate nonubiquitinated aggresome formation. (Left) At 18 h after cotransfection of GFP-250 plasmids with myc-Rheb-Q64L, myc-Rheb-D60K, or vector constructs, COS-7 cells were immunostained for MYC and nuclei (DAPI). (Right) Percentage of aggresome-harboring cells was scored among the cotransfectants as shown. Values represent means ± SD of 3 independent experiments. O/L denotes overlay.

Fig. 3.

Fig. 3.

TSC1 deletion and Rheb activation sensitize misfolded protein-induced apoptosis. (A) TSC1−/− cells are sensitive to MG132. TSC1+/+ and TSC1−/− MEFs with or without 20 nM rapamycin (Rapa) pretreatment for 1 h were challenged with 5 μM MG132 or DMSO for 12 h. Phase-contrast images were taken. (B) MG132 induces apoptosis in TSC1−/− cells. TSC1+/+ and TSC1−/− MEFs with or without 20 nM rapamycin pretreatment for 1 h were challenged with 5 μM MG132 or DMSO for 4 or 9 h before harvest. Cell lysates were blotted for cleaved caspase-3, cleaved PARP, and Akt (for loading control). (C) Rheb-Q64L sensitizes cell death to MG132. (Left) MCF-7 stable cell clones expressing YFP or YFP-Rheb-Q64L treated with 5 μM MG132 for 24 h were immunostained with cleaved caspase-3 antibody, and nuclei were stained with DAPI. (Right) Percentage of cleaved caspase-3 positively stained cells for different clones is shown in diagram.

Fig. 4.

Fig. 4.

TSC1 knockout causes defective aggresome formation and sensitizes misfolded protein-induced apoptosis. (A) TSC1 knockout hepatocytes are compromised in aggresome formation. Shown are representative images of ubiquitin–protein conjugate immunostaining and nuclei staining (DAPI) of liver frozen sections from TSC1flox/flox (F/F) and TSC1flox/flox, albumin-Cre (K/O) littermates with or without (control) PS341 treatment. (B) PS341 induces apoptosis in TSC1−/− liver. PS341 preferentially induces apoptosis in TSC1−/− hepatocytes. Liver homogenates from TSC1flox/flox (F/F) and TSC1flox/flox, albumin-Cre (K/O) littermates with or without (control) PS341 treatment were immunoblotted with TSC1, TSC2, cleaved caspase-3, PARP, and α-tubulin.

Fig. 5.

Fig. 5.

High Rheb activity inhibits the interaction between the dynein motor and ubiquitinated protein cargos. (A) The association between dynein and ubiquitinated proteins is disrupted by TSC1 deletion. TSC1+/+, TSC1−/− MEFs with or without 20 nM rapamycin (Rapa) pretreatment for 1 h were treated with 5 μM MG132 or DMSO for 4 h before harvest. Lysates were normalized and immunoprecipitated (IP) with dynein antibody. Immunoprecipitates were immunoblotted with antibodies for ubiquitin (UB) and dynein. Whole-cell lysates were blotted for P-S6K (T389) and p70 S6K. (B) Rheb knockdown increases cell viability in response to MG132. TSC1 MEFs lentivirally introduced with Rheb shRNA or scramble shRNA were challenged with 5 μM MG132 at 9 h before harvest. RIPA-insoluble cell lysates were normalized and blotted for UB. Soluble lysates were blotted for UB, Rheb, and cleaved caspase-3. (C) A proposed model for TSC1/2–Rheb–mTOR in the regulation of misfolded protein metabolism.

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