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PNAS 106 (22): 8923-8928
Copyright © 2009 by the National Academy of Sciences.
Rheb controls misfolded protein metabolism by inhibiting aggresome formation and autophagy
Xiaoming Zhoua,
Tsuneo Ikenouea,
Xiaowei Chena,
Li Lib,c,
Ken Inokia, and
Kun-Liang Guana,b,c,d,1
aLife Sciences Institute, bDepartment of Biological Chemistry, and dInstitute of Gerontology, University of Michigan, Ann Arbor, MI 48109; and cDepartment of Pharmacology and Moores Cancer Center, University of California at San Diego, La Jolla, CA 92093-0815
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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.
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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- F508. At 18 h after cotransfection of EGFP-CFTRF508 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.
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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.
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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  -tubulin.
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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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