EMBO/soda-nel huggingface dataset:
Cideb controls sterol-regulated ER export of SREBP/SCAP by promoting cargo loading at ER exit sites (2019)
Figure 2

Figure 2. Loss of Cideb protects mice from diet-induced fatty liver diseases.

Liver morphology of WT and Cideb-/- liver under chow diet and HFLF diet. H/E staining, oil red-O staining, immunohistochemistry and ultra-structure (Electron Microscope) were performed. Scale bar represented 50 µm in the upper three rows of images, and 2 µm in the bottom row of images. LD: lipid droplet, ER: endoplasmic reticulum, M: mitochondrial, N: nucleus.

B, C Total liver TAG levels (B) and Liver cholesterol ester levels (C) of WT and Cideb-/- mice under chow diet and HFLF diet (n = 8 per group).

D Loss of Cideb inhibits diet-induced SREBP activation. IB of hepatic SREBP processing and lipogenic enzyme levels (Fasn, Scd1) of WT and Cideb-/- mice under chow diet and HFLF diet.

E Serum AST and ALT levels of WT and Cideb-/- mice under chow diet and HFLF diet (n = 8 per group).

F Cideb deficiency alleviates the whole body inflammation response. Serum TNFα, MCP1 and IL6 levels of WT and Cideb-/- mice under chow diet and HFLF diet (n = 8 per group).

G, H Serum fed glucose levels (G) and insulin levels (H) of WT and Cideb-/- mice under chow diet and HFLF diet (n = 8 per group).

I Glucose tolerance test (GTT) of WT and Cideb-/- mice under HFLF diet (n = 5 per group).

J Insulin tolerance test (ITT) of WT and Cideb-/- mice under HFLF diet (n = 5 per group).

Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.
Cideb controls sterol-regulated ER export of SREBP/SCAP by promoting cargo loading at ER exit sites (2019)
Figure 3

Figure 3. Loss of Cideb specifically inhibits loading of SREBP/SCAP into COPⅡ vesicles.

Decreased SREBP-1/2 and SCAP levels in Golgi apparatus in Cideb-/- mice. IB of organelle fractions of the indicated proteins and quantification of IB data from 3 independent experiments.

Cideb deficiency impairs the packaging of SCAP/SREBP into COPII vesicles. Protein present in the vesicle or microsome fractions obtained from WT or Cideb-/- liver were detected by IB. Quantification of IB data from 3 independent experiments.

Schematic of the BioID assay with Sar1A-BirA*.

Cideb depletion decreases the recruitment of SCAP to the COPII machinery in cells. Primary hepatocytes transfected with control or Cideb siRNA were infected with adenovirus expressing Sar1A-Flag-BirA*. Cells were cultured in sterol-depleted medium and treated with 80 µM biotin, prior to pulldown with streptavidin-conjugated beads. The biotinylated proteins were detected by IB with the indicated antibodies following SDS-PAGE.

Cleaved SREBP bypasses Cideb deficiency. WT and Cideb-/- mice were injected with adenovirus expressing GFP, SREBP-1c full length (FL) or SREBP-1c active form (N). Mice were sacrificed 7 days after injection, and liver TAG levels and hepatic expression SREBP-1 target genes in the indicated groups were determined (n=5 per group).

Data information: Data represent the Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.
Cideb controls sterol-regulated ER export of SREBP/SCAP by promoting cargo loading at ER exit sites (2019)
Figure 4

Figure 4. Sterol-controlled SCAP/Cideb interaction sensitizes SREBP processing.

Cideb co-precipitates with SCAP. Flag-tagged Cideb was co-expressed with different HA-tagged proteins (PLIN2, Insig, SCAP, SREBP-1 or SREBP-2) in 293T cells and immuno-precipitated with an anti-Flag antibody, and the co-immunoprecipitated protein were detected by an anti-HA antibody following SDS-PAGE.

Interaction between the endogenous SCAP and Cideb. Endogenous SCAP was immuno-precipitated from primary hepatocytes under normal medium, 25-HC depletion or addition conditions. And the presence of Cideb in the immune-complex was detected by IB. SREBP-1 was detected as the positive control. Grp94 was detected as the negative control.

Membrane topology of SCAP. The dashed line labels the portion of SCAP (a.a.416-800) that interacts with Cideb.

Cideb interacts with the 416-800 portion of SCAP. Flag-tagged Cideb was co-expressed with the indicated HA-tagged SCAP truncation mutants in 293T cells and immuno-precipitated with an anti-Flag antibody. Levels of the co-immunoprecipitated protein were detected by an anti-HA antibody following SDS-PAGE.

Sterol-dependent interaction switch of SCAP. SCAP interacts with Insig in the presence of sterol, while switches to Cideb binding upon sterol deprivation. Flag-tagged Insig-1 or Cideb was co-expressed with HA-tagged SREBP-2 and SCAP or SCAP-Y298C in CHO-K1 cells in sterol-depleted medium. When indicated, 10 µg/ml 25-HC were supplemented before cells were immuno-precipitated with an anti-Flag antibody, and the levels of co-immunoprecipitated proteins were detected by IB following SDS-PAGE. The dotted blue vertical line was drawn to separate the result between SCAP-WT and SCAP-Y298C.

A "Ping-Pong" scheme of sterol-regulated SCAP/Insig and SCAP/Cideb interactions. SCAP switches from Insig binding to Cideb binding in the absence of sterol, while the sterol-insensitive SCAP-Y298C consistently interacts with Cideb.

Cideb relieves sterol-dependent suppression of SREBP processing. Hepatocytes infected with adenovirus expressing GFP or Flag-Cideb were cultured in sterol deprived condition. When indicated, cells were treated with different doses of 25-HC before subjected to IB with the indicated antibodies following SDS-PAGE. Quantification of IB data from 3 independent experiments. Data represents Mean ± SEM.

Cideb promotes Golgi localization of SCAP. HepG2 cells transfected with GFP-SCAP alone or with HA-Cideb were cultured in sterol deprived conditions or treated with 1 µg/ml 25-HC. Cells were fixed and subjected to confocal microscopy following staining with antibodies against GM130 or HA. Scale bars represent 10 µm (Merge), and 2 µm (Inlay). Bottom: quantification of the percentage of SCAP localized in Golgi-positive region per cell (left) and quantification of the percentage of cells containing Golgi-localized GFP-SCAP (right) under indicated conditions, from 3 independent experiments; Data represent Mean ± SEM; **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.
Cideb controls sterol-regulated ER export of SREBP/SCAP by promoting cargo loading at ER exit sites (2019)
Figure 5

Figure 5. Cideb enhances SREBP processing by interacting with Sec12.

Cideb interacts with Sec12. 293T cells transfected with Cideb and GFP-tagged Sec12, Sec23, Sec16S, or Sec16L were subjected to immuno-precipitation with an anti-Cideb antibody, and levels of the co-immunoprecipitated protein were detected by an anti-GFP antibody.

Co-localization of SCAP and Cideb at the ER exit sites. HepG2 cells transfected with GFP-SCAP and HA-Cideb were arrested with 10 µM BFA and 1 µM Nocodazole for 2 hours, then fixed with methanol and stained with antibodies against Sec12 or HA. 3D reconstruction was obtained by processing 3D-original image with Imaris. Scale bars represent 10 µm (Merge), and 1 µm (Inlay and 3D reconstruction).

Localization of Cideb at the ER exit sites. AML12 cells knocked-in with a GFP tag at the C-terminus of Cideb genome was treated with 10 µM BFA for 1 hour, then fixed with methanol and stained with antibodies against Sec12. Scale bars represent 10 µm (Merge), and 1 µm (Inlay).

Sec12 interacts with the 118-165 portion of Cideb. HA-tagged Sec12 was co-expressed with the indicated Flag-tagged Cideb truncation mutants in 293T cells and immuno-precipitated with an anti-Flag antibody. Levels of the co-immunoprecipitated protein were detected by an anti-HA antibody following SDS-PAGE.

The schematic diagram of Cideb and its truncations.

Sec12/Cideb interaction modelled by molecular dynamic simulation. The red portion highlighted the predicted domain of Cideb interacting with Sec12.

Lysine 128 of Cideb is required for the interaction with Sec12. 293T cells transfected with HA-Sec12 and the indicated Flag-tagged Cideb constructs were subjected to immunoprecipitation with an anti-Flag antibody, and levels of the co-immunoprecipitated HA-Sec12 were detected with an anti-HA antibody.

Recombinant Cideb interacts with Sec12. GST-Sec12 and MBP-tagged Cideb or Cideb-K128A were purified from E.coli and subjected to in vitro binding.

Cideb-K128A fails to promote SREBP processing. Cideb-/- mice were injected with adenovirus expressing GFP, Flag-Cideb, or Flag-Cideb-K128A, and sacrificed 7 days after injection. Liver lysates were subjected to IB with the indicated antibodies following SDS-PAGE and quantification of IB data from 3 independent experiments. Data represent Mean ± SEM; **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.

Cideb, but not Cideb-K128A, promotes SCAP/SREBP incorporation into COPII vesicles. Liver microsomes were isolated from mice expressing the indicated proteins and subjected to in vitro budding assay. Resulting vesicles and the donor microsomes were subject to IB with the indicated antibodies following SDS-PAGE.
Regulation of membrane fluidity by RNF145-triggered degradation of the lipid hydrolase ADIPOR2 (2022)
Figure 1

Figure 1. RNF145 and ADIPOR2 are fatty-acid responsive proteins that localise to the ER.

(A) Fatty-acid induced proteomic changes in HEK-293T cells. (i) Experimental workflow; cells were treated with PA (400 µM), OA (400 µM) or BSA (vehicle control) for 6h in 3 independent experiments and proteomic changes determined by tandem mass tag (TMT)-assisted quantitative mass spectrometry. (ii) Quantitative analysis of proteomic changes in cells treated with PA or OA as described in (i). Proteins with significantly altered abundance (fold change ≥ |1.5|, q ≤ 0.05) are labelled. Proteins involved in lipid metabolism and/or known to be regulated by dietary FAs are indicated in green. RNF145 (bold red) and ADIPOR2 (bold green) were among 'fatty-acid responsive' proteins. N = 3 biological replicates. A summary of the proteomics data is presented in Dataset EV1.

(B) ADIPOR2 is upregulated in RNF145-deficient cells. Quantitative proteomic analysis of HeLa RNF145 knockout (KO) cells versus wild type (WT) (i). ADIPOR2 is indicated in red (ii). Significantly changed proteins (q ≤ 0.05) are highlighted indicated (blue). N = 3 biological replicates.

(C) ADIPOR2 and RNF145 are primarily localised in the endoplasmic reticulum. HEK-293T cells stably expressing tagged constructs were used for immunofluorescence microscopy. (i) Staining of the ER marker protein SP-KDEL-Em (PDI signal peptide fused to mEMERALD-KDEL) and ADIPOR2-S. DNA was stained with DAPI. A magnified view is provided in the tiled micrographs (right). Scale bars = 10 µm / 5 µm (tiles). (ii) RNF45-HAx3 and ADIPOR2-S were stably expressed in UBE2G2-depleted cells and visualised using anti-HA and anti-S tag antibodies. DNA was stained using DAPI. A magnified view is provided in the tiled micrographs (right). Scale bars = 10 µm / 5 µm (tiles).

(D) RNF145 and ADIPOR2 respond to a range of FAs. HEK-293T cells were treated for 6h with saturated FAs (SFAs; 400 µM (C12:0 - C18:0); 200 µM (C20:0)) or unsaturated FAs (UFAs; 400 µM (C18:1-n9); 200 µM (C20:4-n6, C20:5-n3)). * non-specific bands.

(E) Inhibition of the primary lipid desaturase SCD1 affects RNF145 and ADIPOR2 levels. HEK-293T cells were treated with the SCD1 inhibitor MF-438 (SCDi, 100 nM, 18h) and RNF145/ADIPOR2 expression analysed by immunoblotting. * non-specific bands.

(F) SCD1 overexpression affects RNF145/ADIPOR2. HEK-293T cells containing an empty control vector or expressing SCD1-HA were cultured in FCS-free DMEM for 24h and whole-cell lysates analysed. * non-specific bands.
Regulation of membrane fluidity by RNF145-triggered degradation of the lipid hydrolase ADIPOR2 (2022)
Figure 2

Figure 2. Stability of ADIPOR2 is dependent on the activity of RNF145, UBE2G2 and UBXD8.

(A) Endogenous ADIPOR2 is stabilised in RNF145 knockout cells. Representative 35S pulse-chase analysis in HeLa WT, RNF145 knockout (∆RNF145) or ADIPOR2 knockout (∆AR2) cells. Quantification of 35S-labelled ADIPOR2 normalised to t = 0 h is shown below.

(B) ADIPOR2 regulation requires the E3 ligase activity of RNF145. HEK-293T RNF145 knockout cells (∆RNF145) were stably complemented with WT RNF145 (RNF145-V5) or an inactive RNF145C552A,H554A RING domain mutant (mRNF145-V5). * non-specific bands.

(C) RNF145 binds ADIPOR2. RNF145 was immunoprecipitated from HeLa RNF145 knockout cells complemented with WT RNF145 (RNF145-V5) or an inactive RNF145C552A,H554A RING domain mutant (mRNF145-V5). Cells were treated with VCP inhibitor (NMS-873, 10 µM, 90 min) prior to immunoprecipitation with an anti-V5 antibody.

(D) The E2 ubiquitin-conjugating enzyme UBE2G2 is involved in RNF145-mediated ADIPOR2 degradation. Immunoblot analysis of HEK-293T cells depleted of RNF145 (gRNF145), UBE2G2 (gUBE2G2) or B2M (control; gCTR).

(E) ADIPOR2 degradation depends on UBXD8. HEK-293T cells were depleted of B2M (gCTR) or UBXD8 (gUBXD8) and analysed by immunoblotting.
Regulation of membrane fluidity by RNF145-triggered degradation of the lipid hydrolase ADIPOR2 (2022)
Figure 3

Figure 3. ADIPOR2's fatty acid response is modulated by RNF145.

(A) FA-mediated regulation of RNF145 is proteasome-dependent. HEK-293T cells were treated with BSA, PA (400 µM, 6h) ± MG132 (10 µg/ml, 6h) and analysed by immunoblotting. *non-specific bands.

(B) UBXD8 participates in PA-modulated RNF145 degradation. CRISPR/Cas9-mediated depletion of UBXD8 (gUBXD8) or B2M (gB2M, control) in HEK-293T cells, followed by exposure to PA (400 µM, 6h) or BSA (6h, vehicle control).

(C) PA and OA treatment alter RNF145 ubiquitination. Immunoprecipitation of RNF145 from cells treated with BSA (control), PA or OA (each 200 µM, 3h). During FA treatments, VCP inhibitor (NMS-873, 10 µM) was present to stabilise ubiquitinated proteins. RNF145 ubiquitination (ubi) was detected using an anti-ubiquitin antibody (VU-1).

(D) HEK-293T cells were treated with cycloheximide (CHX, 1 µg/ml) and OA (400 µM, 6h) ± MG132 (10 µg/ml) for the indicated times. LE, long exposure; * non-specific bands.

(E) Lysine-less RNF145 is stabilised under PA treatment. RNF145 knockout HEK-293T cells (∆RNF145) were complemented with RNF145-V5 or a lysine-less variant of RNF145 (RNF145-V5 K > R) and treated BSA or PA (400 µM, 6h). Dotted lines demarcate the different cell lines.

(F) Adjustment of RNF145 levels to PA requires its ubiquitin ligase activity. Immunoblot of whole-cell lysates from RNF145 knockout HEK-293T (∆RNF145) cells stably complemented with RNF145-V5 or RNF145C552A, H554A-V5 (mRNF145-V5) and treated with BSA or PA (400 µM, 6h). LE, long exposure.
Regulation of membrane fluidity by RNF145-triggered degradation of the lipid hydrolase ADIPOR2 (2022)
Figure 4

Figure 4. The interaction between RNF145 and ADIPOR2 is FA-sensitive.

(A) ADIPOR2 regulation by FAs is completely dependent on RNF145. WT or RNF145 knockout HEK-293T cells (∆RNF145) were treated with BSA, PA or OA (400 µM, 6h) and analysed by immunoblotting. Non-specific bands are indicated (*).

(B) RNF145 - ADIPOR2 interaction is FA-modulated. Immunoprecipitation of endogenous RNF145 HEK-293T cells depleted of UBE2G2 (gUBE2G2) and/or RNF145 (gRNF145) and treated with 400 µM of the indicated PA, OA or BSA (control) for 3h. LE, long exposure. * non-specific bands.

(C) Immunoprecipitation (IP) of endogenous ADIPOR2 from WT or ∆RNF145 cells (see (C)) treated with MG132 (5 µg/ml, 6h) ± OA (200 µM, 6h). Ubiquitinated ADIPOR2 was detected using a specific anti-ubiquitin antibody (clone VU-1). Note that ADIPOR2 levels in RNF145 knockout HEK-293T cells are increased.

(D) ADIPOR2 knockout HEK-293T cells were stably complemented with ADIPOR2-HA, an ADIPOR2 mutant in which all lysines were mutated to arginines (K-less ADIPOR2-HA), or an empty vector control and exposed to OA (200 µM, 6h) or BSA (control). ADIPOR2 variants were visualised by immunoblotting using a specific anti-HA antibody. LE, long exposure.

(E) Model of RNF145-mediated ADIPOR2 degradation. RNF145 uses UBE2G2 to target ADIPOR2 for UBXD8/VCP-mediated extraction from the ER membrane and subsequent proteasomal degradation. PA decreases RNF145 binding to ADIPOR2, while OA promotes ADIPOR2's interaction with and ubiquitination by RNF145 (white arrows).
Inhibition of lethal inflammatory responses through the targeting of membrane-associated Toll-like receptor 4 signaling complexes with a Smad6-derived peptide. (2015)
Figure 1

Amino acids 400–441 of Smad6 specifically bind to Pellino-1 and inhibit NF-κB signaling

A Schematic representation of truncated mutants of the Smad6 MH2 domain and binding to Pellino-1N. HA-tagged Pellino-1N encodes amino acids 1–137 of Pellino-1.

B Plasmids encoding truncated mutants of the Smad6 MH2 domain were co-transfected into HEK293 cells with HA-tagged Pellino-1N plasmid. Cell lysates were immunoprecipitated with anti-HA antibody and immunoblotted with anti-Myc antibody. Data are representative of at least three independent experiments. IP, immunoprecipitation; IB, immunoblot; TCL, total cell lysates.

C, D The SBE-Luc or 5xNF-κB-Luc reporter plasmid was co-transfected with an empty vector, Myc-Smad6 (400–441), or full-length Smad6-expressing plasmids into CMT-93 cells, respectively. After 24 h, cells were treated with TGF-β1 for 2 h or LPS for 2 h and luciferase activities were measured and normalized. Data were statistically analyzed by a t-test and show the mean ± SD of three independent experiments. **P < 0.05, ***P < 0.001.

E CMT-93 cell lines stably expressing Smad6 amino acids 400–441 were treated with LPS for the indicated time and expression of IκBα, IKKα, and phospho-IKKα/β was monitored by immunoblotting. As a positive control, CMT-93 cells were pre-treated with TGF-β1 for 2 h. CMT-93 cells stably expressing the empty vector pMSCV-puro were used as a negative control. β-actin was used as a loading control. All data are representative of three independent experiments.

Source data are available online for this figure.
Inhibition of lethal inflammatory responses through the targeting of membrane-associated Toll-like receptor 4 signaling complexes with a Smad6-derived peptide. (2015)
Figure 2

The minimal Pellino-1-binding region of Smad6 selectively inhibits NF-κB signaling

A Schematic representation of truncated mutants of the Smad6 MH2 domain.

B A plasmid encoding the truncated mutant composed of Smad6 amino acids 422–441 (Myc-Smad6(422-441)) or wild-type Smad6 MH2 domain (Myc-Smad6-MH2) was co-transfected with the HA-tagged full-length Pellino-1 plasmid into HEK293 cells. Cell lysates were immunoprecipitated (IP) with anti-Myc or anti-HA antibody, and immunoblotted (IB) with anti-HA or anti-Myc antibody, respectively. The vector, CS3MTBXA-6xMyc, was transfected as a negative control. IP, immunoprecipitation; IB, immunoblot; TCL, total cell lysates. Data are representative of at least three independent experiments.

C The Myc-Smad6(422-441) plasmid was co-transfected with full-length Flag-IRAK1, Flag-TRAF6, Flag-MyD88, or HA-Pellino-1 plasmid into HEK293 cells. Cell lysates were immunoprecipitated with the indicated antibodies and immunoblotted with anti-Myc antibody. IgG was added as a negative control for IP. Data are representative of at least three independent experiments.

D, E The SBE-Luc or 5xNF-κB-Luc reporter plasmids were co-transfected with an empty vector or the Myc-Smad6(422-441) plasmid into CMT-93 cells, respectively. After 24 h, cells were treated with TGF-β1 for 6 h or LPS for 2 h, and luciferase activities were measured and normalized.

F The BRE-Luc reporter plasmid was co-transfected with an empty vector or the Myc-Smad6(422-441) plasmid or full-length Smad6 into RAW264.7 cells, respectively. After 24 h, cells were treated with BMP4 for 6 h and luciferase activity was measured and normalized.

G A plasmid encoding the truncated mutant composed of Smad6 amino acids 422–441 (Myc-Smad6(422-441)) or wild-type Smad6 MH2 domain (Myc-Smad6-MH2) or full-length Smad6 (Myc-Smad6) was co-transfected with HA-tagged full-length Smad4 or HA-tagged full-length Pellino-1 plasmid into HEK293 cells, respectively. Cell lysates were immunoprecipitated (IP) with anti-Myc and immunoblotted (IB) with anti-HA or anti-Myc antibody, respectively. The vector, pCS3MTBXA-6xMyc, was co-transfected with HA-Smad4 or HA-Pellino-1 as a negative control. Data are representative of at least three independent experiments.

Data information: All data in (D–F) were statistically analyzed by a t-test and show the mean ± SD of three independent experiments. **P < 0.005, *P < 0.05 compared to the control (without LPS, TGF-β1, or BMP4).

Source data are available online for this figure.
Inhibition of lethal inflammatory responses through the targeting of membrane-associated Toll-like receptor 4 signaling complexes with a Smad6-derived peptide. (2015)
Figure 3

Membrane-tethered Smaducin-6 inhibits TLR4 signaling

A Pre-treatment of Smaducin-6 reduces LPS-induced interleukin-6 (Il6) gene expression in a dose-dependent manner in RAW264.7 cells. Il6 gene expression was analyzed by quantitative real-time RT–PCR.

B, C Pre-treatment of 100 nM Smaducin-6 or scrambled peptide (Pal-Scram #1) for 30 min inhibits (B) NF-κB-mediated luciferase gene expression and (C) IκBα degradation and IKKα/β phosphorylation when RAW264.7 cells are treated with LPS for 2 h. Luciferase activity in (B) was normalized to β-galactosidase activity.

D Peli1 knockdown or wild-type human THP1 cells were pre-treated with 100 nM Pal-Scram peptide and Smaducin-6 for 30 min and subsequently treated with LPS for 2 h. Il6 and Peli1 gene expression were analyzed by quantitative real-time RT–PCR. Data show the mean ± SD of three independent experiments.

E RAW264.7 cells were treated with 100 nM FITC-conjugated Smaducin-6 and TAT-S6(422-441), and localization was observed by confocal microscopy. Scale bar, 10 μm (magnification, 200×). Nuclei were stained with DAPI.

F After pre-treating RAW264.7 cells with biotin-conjugated Smaducin-6 (100 nM) for 30 min, cells were treated with LPS for 2 h. Subsequent precipitation by streptavidin–agarose showed that endogenous Pellino-1 binds to the Smaducin-6 peptide. A biotin-conjugated scrambled peptide was used as a negative control.

G–I Immunoprecipitation assays in primary peritoneal macrophages show that Smaducin-6 inhibits the formation of endogenous (G) IRAK1-, (H) RIP1-, or (I) IKKε-mediated signaling complexes induced by 2 h LPS treatment, compared to a scrambled peptide. IB, immunoblot; TCL, total cell lysates. Data shown are representative of three independent experiments.

Data information: Data in (A) and (B) were statistically analyzed by a t-test and show the mean ± SD of three independent experiments. ***P < 0.001, **P < 0.005, *P < 0.05 compared to no LPS treatment or vehicle control (Pal-Scram #1).

Source data are available online for this figure.