Model for CHIP regulation of iNOS degradation and aggresome formation. CHIP interaction with iNOS is mediated by CHIP's chaperone-binding domain. CHIP's U-box domain recruits the E2 ubiquitin conjugation enzyme and ubiquitinates iNOS. Ubiquitinated iNOS is targeted for proteasomal degradation and to aggresome sequestration. The latter mechanism is accelerated by proteasomal inhibition. CHIP-induced iNOS ubiquitination promotes association of iNOS to HDAC6, which serves as a linker between ubiquitinated iNOS and the dynein motor. Dynein motor-dependent microtubular transport moves iNOS to the aggresome.

Ubiquitin ligase activity is required for CHIP's role in aggresome formation. HEK293 cells were transfected for 48 h with lentivirus expressing either control shRNA or shRNA specific for CHIP. Cells were then transfected for 24 h with plasmids expressing either iNOS-GFP (A) or GFP-250 (B) and incubated for 3 h in the presence of a 10 μM concentration of the proteasome inhibitor MG132. Cells were then immunostained with ubiquitin or CHIP antibodies. Scale bar, 10 μm.

Ubiquitin ligase activity of CHIP is required for aggresome formation. HEK293 cells were cotransfected with a plasmid encoding the cDNA of human iNOS and with either a plasmid encoding WT CHIP (row i) or CHIP-H260Q. The molar ratio of the iNOS plasmid to the coplasmid was 1:5. Forty-eight hours after transfection, cells were fixed and immunostained with antibodies against His-tagged CHIP, ubiquitin, or γ-tubulin. Graphs show quantitation of the percentage of cells expressing iNOS aggresome and preaggresome structures. Data represent means ± standard deviations (n = 3). *, P < 0.05; **, P < 0.001 (compared to control condition). Scale bar, 10 μm.

Disruption of dynein-dependent microtubular transport leads to accumulation of preaggresomal structures and prevents iNOS aggresome formation. HEK293 cells stably expressing iNOS-GFP were fixed and analyzed by fluorescence microscopy. (A) Cells were pretreated with DMSO (vehicle) or 1 μM nocodazole for 1 h, followed by either vehicle or 1 μM nocodazole plus 10 μM MG132 for 3 h. (B) Experiments were done as in panel A, except that 1 mM EHNA was used instead of nocodazole. (C) Cells were transfected with control vector or Flag-tagged p50. Twenty-four hours after transfection, cells were treated with 10 μM MG132 for 4 h, fixed, and immunostained with flag antibody.

CHIP promotes iNOS proteasomal degradation. HEK293 cells were cotransfected with human iNOS cDNA and with either a control vector or with CHIP in a 1:5 molar ratio. At 24 h posttransfection, cells were pulsed with [³⁵S]methionine-cysteine for 1 h and chased with unlabeled medium at various time points in the absence (A) or the presence (B) of a 10 μM concentration of the proteasome inhibitor MG132. iNOS was immunoprecipitated with iNOS antibody, eluted, and analyzed by SDS-PAGE. Bands representing ³⁵S-labeled iNOS were quantitated to calculate the iNOS half-life. Data represent means ± standard deviations (n = 3). *, P < 0.05.

CHIP colocalizes with iNOS aggresome. (A) HEK293 cells were cotransfected with a plasmid encoding the cDNA of iNOS-GFP and with a plasmid containing a vector as a control or Flag-tagged CHIP. The molar ratio of the iNOS plasmid to the coplasmid was 1:10. Forty eight hours after transfection, cells were fixed and examined by fluorescence microscopy. Exogenously expressed CHIP colocalized with cytosolic iNOS (row i) and translocated with iNOS to the aggresome (row ii). iNOS aggresomes in CHIP-transfected cells were ubiquitin enriched (row iii). (B) Endogenous CHIP colocalized with the iNOS aggresome in primary airway epithelial cells cultured at the air-liquid interphase (row i), in RT4 cells (row ii), and in HEK293 cells (row iii). Primary cells and RT4 cells expressed iNOS following cytokine stimulation, whereas HEK293 cells stably expressed iNOS-GFP. Scale bar, 10 μm.

Knockdown of CHIP prevents aggresome formation by CFTRΔF508. HEK293 cells were transfected for 48 h with plasmids expressing either control shRNA or shRNA specific for CHIP. Cells were then transfected for 24 h with a plasmid expressing CFTRΔF508-GFP. Cells were incubated for 6 h in the presence of vehicle only (DMSO) (A) or a 10 μM concentration of the proteasome inhibitor MG132 (B). Cells were then fixed, stained with DAPI to visualize the nuclei (blue), immunostained with CHIP antibody, and examined by fluorescence microscopy. Arrows point to the mature CFTRΔF508 aggresome. Graphs show quantitation of the percentage of cells expressing CFTRΔF508 aggresome or preaggresome structures. Data represent mean ± standard deviation (n = 2). *, P < 0.05, compared to control condition. Scale bar, 10 μm.

Knockdown of endogenous CHIP increases iNOS cellular level. (A). HEK293 cells stably expressing iNOS were transfected for 72 h with a plasmid encoding either control shRNA having no perfect matches to any human gene or shRNA specific for CHIP. (B) RT4 cells were transduced with lentivirus encoding CHIP-specific shRNA or control shRNA. After 48 h, cells were incubated with gamma interferon (100 U/ml), interleukin-1β (0.5 ng/ml), and tumor necrosis factor alpha (10 ng/ml) for 24 h. In both panels, cells were lysed, and Western analysis was used to evaluate CHIP, iNOS, or GAPDH. iNOS activity was evaluated by measuring nitrite accumulation in the culture medium. Data represent mean ± standard deviation. n = 3 (A) or 4 (B). *, P < 0.05, compared to the control condition.

Critical role for CHIP in iNOS aggresome formation. (A and B) HEK293 cells stably expressing iNOS-GFP were transfected for 72 h with plasmids expressing either control shRNA or shRNA specific for CHIP. Cells were incubated for 3 h in the presence of vehicle only (DMSO) (A) or a 10 μM concentration of the proteasome inhibitor MG132 (B). Cells were then fixed, stained with DAPI to visualize the nuclei (blue), immunostained with CHIP antibody, and examined by fluorescence microscopy. Arrows in panel B point to the mature iNOS aggresome. Graphs show quantitation of the percentage of cells expressing iNOS aggresome or preaggresome structures. (C to E) HEK293 cells were cotransfected with a plasmid encoding cDNA of human iNOS-GFP and with a plasmid containing vector as a control or Flag-tagged CHIP. The molar ratio of the iNOS plasmid to the coplasmid was 1:10. At 24 (C) or 48 (D and E) h after transfection, cells were examined by fluorescence microscopy. Quantitation of the percentage of cells expressing the iNOS aggresome is shown (C and D). CHIP-transfected cells exhibited larger iNOS aggresomes (E). Data represent mean ± standard deviation (n = 3). *, P < 0.05; **, P < 0.001 (compared to control condition). Scale bar, 10 μm.

CHIP promotes HDAC6-mediated iNOS aggresome formation. (A) CHIP increases the association of iNOS with HDAC6. HEK293 cells were cotransfected for 24 h with a plasmid encoding iNOS and with a plasmid containing control vector, WT CHIP, the CHIP-H260Q mutant, or the CHIP-K30A mutant. The molar ratio of the iNOS plasmid to the coplasmid was 1:1. Coimmunoprecipitation was carried out on cell lysates using antibodies against iNOS or HDAC6. Immunoprecipitated proteins were analyzed by Western blotting for the presence of iNOS or HDAC6. Immunoprecipitation (IP) with irrelevant antibody (IκB-β) was used as a negative control. Western analysis (lower panel) of cell lysates was done prior to immunoprecipitation using equal aliquots corresponding to 2.5% of amount of proteins used for immunoprecipitation. (B) Knockdown of CHIP decreases the interaction of iNOS and HDAC6. HEK293 cells were transduced for 48 h with lentiviral vector expressing either control shRNA or CHIP shRNA and then transfected with iNOS cDNA for 24 h. Coimmunoprecipitation (IP) and Western analysis were done as described in panel A. (C) HDAC6 colocalizes with the iNOS aggresome but not with cytosolic iNOS or preaggresomal structures caused by CHIP-H260Q. (i) HEK293 cells stably expressing iNOS-GFP without aggresome formation. (ii) Cells exhibiting iNOS aggresome following MG132 treatment. (iii) Cells with iNOS aggresome promoted by overexpression of WT-CHIP. (iv) Cells with iNOS preaggresomal structures promoted by overexpression of CHIP-H260Q. (D) Knockdown of HDAC6 prevents iNOS aggresome formation. HEK293 cells stably expressing iNOS-GFP were transfected with vector encoding control shRNA or shRNA specific for HDAC6 for 3 days followed by another 3-day transfection with the same vectors to ensure knockdown of HDAC6. Western analysis of cell lysates was performed with antibodies against HDAC6, iNOS, acetylated tubulin, or tubulin (upper panel). Cells were treated with 10 μM MG132 for 10 h, fixed, and analyzed by immunofluorescence with HDAC6 antibody (middle panel). The graph shows quantitation of the percentage of cells expressing iNOS aggresome or preaggresome structures. Data represent means ± standard deviations (n = 3). *, P < 0.05. Scale bar, 10 μm.

Contributions of the chaperone binding domain and ubiquitin ligase domain to CHIP's role in cellular triage of iNOS. (A) Diagram and domain organization of CHIP. TPR-containing domain mediating interaction with chaperones (violet) and E3 ubiquitin ligase domain (green; U-box) are highlighted. The mutation sites of the chaperone binding-deficient mutant CHIP-K30A and the ubiquitin ligase-deficient mutant CHIP-H260Q are shown. HEK293 cells were transfected for 24 h with WT CHIP or CHIP mutants. Cells were lysed, and Western analysis was carried out to evaluate CHIP or GAPDH using His-tagged antibody or GAPDH antibody, respectively. (B) CHIP-H260Q mutant inactivates iNOS. HEK293 cells were cotransfected for 24 h with a plasmid encoding cDNA of human iNOS and with a plasmid containing control vector, WT His-CHIP, the His-CHIP-H260Q mutant, or the His-CHIP-K30A mutant. The molar ratio of the iNOS plasmid to the coplasmid was 1:5. iNOS activity was evaluated by measuring nitrite accumulation in culture medium (mean ± standard deviation; n = 6). (C) CHIP-H260Q mutant exhibits avid interaction with iNOS. HEK293 cells were cotransfected as in panel B except that the molar ratio of the iNOS plasmid to the coplasmid was 1:1. Twenty-four hours after transfection, cells were incubated with a 10 μM concentration of the proteasome inhibitor MG132 for 6 h and then lysed in NETN buffer. Cell lysates were subjected to immunoprecipitation (IP) with iNOS antibody or with His-tagged antibody. An aliquot of the immunoprecipitate was subjected to Western blotting with iNOS antibody or with His-tagged antibody. Immunoprecipitation with IκB-β antibody was used as a negative control. Western analysis (lower panel) was done on cell lysates prior to immunoprecipitation. Protein loading was done in equal aliquots corresponding to 2.5% of the amount of proteins used for immunoprecipitation. (D) CHIP-H260Q mutant does not ubiquitinate iNOS. HEK293 cells were cotransfected as in panel B. At 24 h posttransfection, cells were incubated with 10 μM MG132 for 3 h and then lysed. Cell lysates were subjected to immunoprecipitation (IP) with an iNOS antibody. An aliquot of the immunoprecipitate was subjected to Western blotting (W) with ubiquitin (Ub) antibody. (E and F) HEK293 cells were cotransfected as in panel B. At 24 (E) or 48 (F) h after transfection, cells were lysed using 1% Triton X-100 as a detergent, and lysates were divided into Triton X-100-soluble (S) and Triton X-100-insoluble (IS) fractions. Equal aliquots of cell lysates (50 μg) were evaluated by Western blotting using antibodies against iNOS, CHIP, or GAPDH. *, P < 0.05; **, P < 0.001.