Regulation of Cidea protein stability by the ubiquitin-mediated proteasomal degradation pathway

Abstract

Cidea, one of three members of the CIDE (cell-death-inducing DNA-fragmentation-factor-45-like effector) family of proteins, is highly enriched in brown adipose tissue, in which it plays a critical role in adaptive thermogenesis and fat accumulation. Cidea-null mice have increased energy expenditure with resistance to high-fat-diet-induced obesity and diabetes. However, little is known as to how the Cidea protein is regulated. In the present study we show that Cidea is a short-lived protein as measured by cycloheximide-based protein chase experiments in different cell lines or in differentiated brown adipocytes. Proteasome inhibitors specifically increased the stability of both transfected and endogenous Cidea protein. Furthermore, Cidea protein was found to be polyubiquitinated when overexpressed in different culture cells as well as in differentiated mature brown adipocytes. Extensive mutational analysis of individual lysine residues revealed that ubiquitinated lysine residues are located in the N-terminal region of Cidea, as alteration of these lysine residues to alanine (N-5KA mutant) renders Cidea much more stable when compared with wild-type or C-terminal lysine-less mutant (C-5KA). Furthermore, K23 (Lys23) within the N-terminus of the Cidea was identified as the major contributor to its polyubiquitination signal and the protein instability. Taken together, the results of our study demonstrated that the ubiquitin-proteasome system confers an important post-translational modification that controls the protein stability of Cidea.

Figure 1. Cidea is a short-lived protein and its degradation is proteasome-dependent

(A, B and C) A 1 μg portion of CMV5–HA–hCidea was co-transfected with 0.5 μg of pEGFP-N1 into HEK-293T, CHO-K1 and H1299 cells in 60-mm-diameter dishes using Dosper reagent. CHX-based protein chase experiments were performed as described below. At 24 h post-transfection, and 1 h prior to the addition of CHX, the medium was replaced with fresh medium, and then CHX was added to a final concentration of 100 μg/ml. Cells were harvested in 0.5 ml of lysis buffer at different time points: 0, 30, 60 and 120 min. Total cell lysates were prepared and analysed by Western blotting [IB (immunoblotting)] using anti-HA and anti-GFP as primary antibodies. The amount of GFP transiently co-expressed by pEGFP-N1 present in total cell lysates was used for the normalization of transfection protein. (D) Graph showing band intensities corresponding to HA–Cidea and GFP scanned and quantitified using ImageQuant-TL software (Amersham). Results are expressed as the percentage of the signal of HA-Cidea compared with that of GFP. The ratio was set to 100% at zero time. The results shown are representative of three independent experiments. (E) HEK-293T cells in 60-mm-diameter dishes were co-transfected with 1 μg of CMV-HA vector (lane 1) or 1 μg of CMV5–HA–hCidea (lane 2–11) and 0.5 μg of pEGFP-N1 using the calcium phosphate method. After 24 h, CHX was added to 100 μg/ml (lanes 3–11) along with different reagents [DMSO, ethanol, pepstatin (0.5 μg/ml), leupeptin (5 μg/ml), aprotinin (2 μg/ml), ALLN (50 μM), MG132 (10 μM), chloroquine (50 μg/ml) and NH₄Cl (2.5 mM)] for 2 h. Cells were harvested in 0.5 ml of lysis buffer after the 2 h treatment. Total cell lysates were subjected to Western blotting and detected with anti-HA and anti-GFP antibodies.

Figure 2. Endogenous Cidea in differentiated brown adipocytes is also unstable and proteasome inhibitor MG132 can prevent its degradation

Brown preadipocytes, obtained by isolation of fresh BAT from 4-week-old Balb/c mice, were grown to confluency in 100-mm-diameter tissue-culture dishes and maintained in DMEM plus 10% FBS. After that, brown preadipocytes were allowed to differentiate into mature adipocytes in the presence of 0.5 mM IBMX and 10 μM dexamethasone at day 0. Then, at day 2, 48 h later, the differentiation medium was replaced with DMEM plus 10% FBS, 1 μg/ml insulin and 10 μM pioglitazone. The differentiation medium was continually changed each day until day 8. At 2 h before treatment with drugs, the medium of differentiated brown mature adipocytes was replaced with fresh medium without the differentiation agents. DMSO, 100 μg/ml CHX and 10 μM MG132 were added for 2 h, and 0.5 ml portions of cell extract were harvested in lysis buffer for Western-blot analysis using anti-Cidea, anti-UCP1 and anti-β-tubulin antibodies. (A) Lane 1, undifferentiated cells from primary preadipocytes; lanes 2 and 3, differentiated mature brown adipocytes treated with control DMSO and CHX for 2 h. (B) Lane 1, undifferentiated cells from primary preadipocytes; lanes 2 and 3, differentiated mature brown adipocytes treated with control DMSO and MG132 for 2 h. IB, immunoblotting.

Figure 3. Cidea can be ubiquitinated by exogenous or endogenous ubiquitin

(A) HEK-293T cells in 60-mm-diameter dishes were co-transfected with 0.5 μg of pXJ-40-HA-Ub, pEGFP-N1 and 1 μg of CMV5–Flag control vector or CMV5–Flag–hCidea using the calcium phosphate method. In vivo ubiquitination was determined as follows. At 1 h before treatment with MG132, the medium was replaced with fresh medium and then MG132 was added to a final concentration of 10 μM for 2 h. Then cells were harvested in 0.5 ml of lysis buffer containing 0.5% SDS, 5 mM dithiothreitol and heated at 90 °C for 5 min, 0.25 ml of sonicated and centrifuged lysate was then subjected to IP (immunoprecipitation) by diluting SDS to 0.1% in lysis buffer using M2 beads, and Western-blotting analysis was conducted using anti-ubiquitin, anti-Flag and anti-GFP antibodies. (B) HEK-293T cells in 60-mm-diameter dishes were co-transfected with 1 μg of CMV5–HA control vector or CMV5–HA–Cidea and 0.5 μg of pEGFP-N1 using the calcium phosphate method. At 24 h post-transfection, MG132 was added to a final concentration of 10 μM for 2 h (lanes 2 and 3) as indicated. Then cells were harvested using the same procedure as for the in vivo ubiquitination assay as described above and were subjected to IP using anti-HA antibody and Protein A/G Plus–agarose beads. The immunoprecipitates and total cell lysates were analysed by Western blotting using anti-ubiquitin, anti-HA and anti-GFP antibodies. (C) One 100-mm-diameter dish of differentiated mature adipocytes from the HB2 cell line at day 8, 2 h prior to treatment with MG132, the medium was replaced with fresh medium and then MG132 (10 μM) was added for 2 h. After MG132 treatment, cells were harvested in lysis buffer and lysates prepared were subjected to IP with anti-Cidea antibody (lanes 1 and 2) and control IgG (lane 3) for 4 h as indicated. The IP product was washed in lysis buffer four times and finally dissolved in 30 μl of lysis buffer. The freshly boiled samples were detected by Western blotting using anti-ubiquitin, anti-Cidea and anti-β-tubulin antibodies. IB, immunoblotting.

Figure 4. Cidea N-terminal lysine residues play a predominant role in controlling protein stability

(A) Schematic drawing of human Cidea, WT, N-5KA, C-5KA and KO mutants. (B) A 1.0 μg portion of CMV5–HA–Cidea, CMV5–Cidea–N-5KA, CMV5–Cidea–C-5KA and CMV5–Cidea-KO were co-transfected with 0.5 μg of pEGFP-N1 in HEK-293T cells in 60-mm-diameter dishes using the calcium phosphate method. The CHX-based protein chase experiment was performed as described above. Total cell lysates were detected by Western blotting using anti-HA and anti-GFP antibodies. The blot shown is representative of three separate experiments. (C) Quantification of scanned gel bands shown in (B) was performed using ImageQuant-TL software (Amersham). Results are expressed as the percentage of the signal of HA-Cidea compared with that of GFP. The value was set to 100% at zero time. The results shown are representative of the three independent experiments. (D) HEK-293T cells in 60-mm-diameter dishes were co-transfected with 1.0 μg of CMV5-HA control vector, CMV5–HA–Cidea (WT), CMV5–HA–Cidea–N-5KA and CMV5–HA–Cidea–C-5KA or CMV5–HA–Cidea-KO control vector with 0.5 μg of pXJ-40-Myc-Ub and pEGFP-N1. Then in vivo ubiquitination assays were performed as described above and cell lysates were subjected to IP using anti-HA in protein A/G beads. The immunoprecipitates were analysed by Western blotting using anti-Myc, anti-HA and anti-GFP antibodies. (E) Apoptosis induced by Cidea and its lysine-less mutants. A 1.5 μg portion of the indicated plasmids and 0.5 μg of pEGFP-N1 were co-transfected into CHO-K1 cells. Apoptotic cells were quantified, and the percentage of cell death was calculated retative to the GFP-positive cells. Results are means±S.D. for at least three independent experiments. IB, immunoblotting.

Figure 5. Cidea K23 is the major contributor to protein instability

(A) Amino acid sequence alignment of Cidea sequence in mammals using ClustalX (version 1.83) software. The shaded regions indicate identical amino acids and conserved lysine residues. The proposed Cidea Cide-N domains are overlined. (B) A 1.0 μg portion of CMV5–HA vector, CMV5–HA–Cidea and CMV–HA–Cidea–K23A, CMV–HA–Cidea–K23E, CMV–HA–Cidea–K23G or CMV–HA–Cidea–K23R was co-transfected with 0.5 μg of pEGFP-N1 in HEK-293T cells in 60-mm-diameter dishes using the calcium phosphate method. The CHX-based protein chase experiment was performed as described above. Total cell lysates were detected by Western blotting using anti-HA and anti-GFP antibodies. The blot shown is representative of three separate experiments. (C) Quantification of scanned gel bands shown in (B) was performed using ImageQuant-TL software (Amersham) and results are expressed as the percentage of the signal of HA-Cidea compared with that of GFP. The ratio was set to 100% at zero time. The results shown are representative of three independent experiments. (D) HEK-293T cells were co-transfected with 1.0 μg of CMV5–HA control vector, CMV5–HA–Cidea, CMV5–HA–Cidea–K23A, CMV–HA–Cidea–K23E, or CMV–HA–Cidea–K23G or CMV–HA–Cidea–K23R with 0.5 μg of pXJ-40-Myc-Ub and pEGFP-N1. Then an in vivo ubiquitination assay was performed as described above and cell lysates were subjected to IP (immunoprecipitation) using anti-HA and Protein A/G Plus–agarose beads. The immunoprecipites were analysed by Western blotting using anti-Myc, anti-HA and anti-GFP antibodies. IB, immunoblotting.