Journal: iScience

Article Title: The protease DDI2 regulates NRF1 activation in response to cadmium toxicity

doi: 10.1016/j.isci.2022.105227

Figure Lengend Snippet: Cadmium indirectly inhibits the proteasome (A) Relative proteasome activity of HepG2 cells treated with CdCl 2 (Cd; + 1 μM or ++ 5 μM) or carfilzomib (CFZ, 10 nM) during 6 h. Each bar represents the mean, and error bars denote SD of at least three independent experiments. (B) Relative proteasome activity of purified 26S proteasome complexes incubated with the substrate Suc-LLVY-AMC and treated with or without Cd (1 μM) or CFZ (10 nM) during the indicated time in minutes (n = 3).

Article Snippet: Purified human proteasome complexes , Bio-Techne , E−365-025.

Techniques: Activity Assay, Purification, Incubation

Journal: iScience

Article Title: The protease DDI2 regulates NRF1 activation in response to cadmium toxicity

doi: 10.1016/j.isci.2022.105227

Figure Lengend Snippet:

Article Snippet: Purified human proteasome complexes , Bio-Techne , E−365-025.

Techniques: Recombinant, Purification, Bicinchoninic Acid Protein Assay, Activity Assay, MTS Assay, Expressing, Western Blot, Plasmid Preparation, Software

Journal: Scientific Reports

Article Title: The Cullin 4A/B-DDB1-Cereblon E3 Ubiquitin Ligase Complex Mediates the Degradation of CLC-1 Chloride Channels

doi: 10.1038/srep10667

Figure Lengend Snippet: Biochemical inspection of CLC-1 protein interactions. (A) Co-immunoprecipitation of HA-CLC-1 and Myc-CUL4A/B in HEK293T cells. Co-expression of HA-CLC-1 and the Myc vector was used as the control experiment. (B) Co-immunoprecipitation of Myc - CLC-1 and Flag-DDB1 in HEK293T cells. Co-expression of Flag-DDB1 and the Myc vector was used as the control experiment. (C) Lack of co-immunoprecipitation of Myc-CLC-1 and Flag-DDB2 in HEK293T cells. Co-expression of Flag-DDB2 and the Myc vector was used as the control experiment. (D) Co-immunoprecipitation of Myc - CLC-1 and HA-CRBN in HEK293T cells. Co-expression of Myc-CLC-1 and the HA vector was used as the control experiment. Cell lysates were immunoprecipitated with the anti-Myc ( A,B,C ) or anti-HA ( D ) antibody. (E) Protein interactions of endogenous CLC-1 channels in rat skeletal muscle. Verification of the specificity of the anti-CLC-1 antibody: ( Far left ) CLC-1 immunoreactivity was notably reduced in the presence of an excess of a control antigen peptide; ( Center left ) immunoprecipitation was achieved by using the anti-CLC-1 antibody, but not rabbit IgG. ( Center right , Far right ) Co-immunoprecipitation of CLC-1, CRBN, DDB1, and CUL4B. Muscle lysates were immunoprecipitated with the anti-CLC-1 antibody. h.c.: IgG heavy chain. The gels were run under the same experimental conditions. Uncropped images of immunoblots are shown in .

Article Snippet: Other cDNA constructs employed in this study include pcDNA3.1-Flag dominant-negative human cullin 1/2/3/4A/4B/5 (Addgene 15818-15823), pcDNA3-Myc human cullin 3/4A/4B (Addgene 19893, 19951, 19922), pcDNA3-HA wild-type and lysine-less human ubiquitin (kindly provided by Dr. Chihiro Sasakawa, University of Tokyo, Japan), pcDNA3-Flag human DDB1 (Addgene 19918), pcDNA3-Flag human DDB2 (kindly provided by Dr. Show-Li Chen, National Taiwan University, Taiwan), and pcDNA3-HA rat cereblon (kindly provided by Dr. Chul-Seung Park, Gwangju Institute of Science and Technology, Korea).

Techniques: Immunoprecipitation, Expressing, Plasmid Preparation, Control, Western Blot

Journal: Scientific Reports

Article Title: The Cullin 4A/B-DDB1-Cereblon E3 Ubiquitin Ligase Complex Mediates the Degradation of CLC-1 Chloride Channels

doi: 10.1038/srep10667

Figure Lengend Snippet: Biochemical demonstration of the regulation of CLC-1 by CRBN in HEK293T cells. (A) (Top) Representative immunoblots showing the effect of Flag-DDB1 or HA-CRBN over-expression on Myc-CLC-1 protein. Co-expression with the Flag/HA vector was used as the control experiment. (Bottom) Quantification of relative CLC-1 protein expression level. Values from the DDB1/CRBN co-expression group ( hatched bars ) were normalized to those for the corresponding vector control ( clear bars ). Asterisks denote significant difference from the control (*, t -test: p < 0.05; n =6-12). (B) CLC-1 polyubiquitination [CLC-1-(Ub)n] by endogenous ubiquitin was enhanced by CRBN over-expression. Cell lysates were immunoprecipitated (IP) with the anti-Myc antibody. Protein ubiquitination was identified by immunoblotting the immunoprecipitates with the anti-ubiquitin (Ub) antibody. (C) Representative immunoblots showing the effect of shRNA knock-down of endogenous DDB1 or CRBN. The numbers denote the relative CLC-1/DDB1/CRBN expression level with respect to the control shRNA for GFP. The gels were run under the same experimental conditions. Uncropped images of immunoblots are shown in .

Article Snippet: Other cDNA constructs employed in this study include pcDNA3.1-Flag dominant-negative human cullin 1/2/3/4A/4B/5 (Addgene 15818-15823), pcDNA3-Myc human cullin 3/4A/4B (Addgene 19893, 19951, 19922), pcDNA3-HA wild-type and lysine-less human ubiquitin (kindly provided by Dr. Chihiro Sasakawa, University of Tokyo, Japan), pcDNA3-Flag human DDB1 (Addgene 19918), pcDNA3-Flag human DDB2 (kindly provided by Dr. Show-Li Chen, National Taiwan University, Taiwan), and pcDNA3-HA rat cereblon (kindly provided by Dr. Chul-Seung Park, Gwangju Institute of Science and Technology, Korea).

Techniques: Western Blot, Over Expression, Expressing, Plasmid Preparation, Control, Ubiquitin Proteomics, Immunoprecipitation, shRNA, Knockdown

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Inositol hexakisphosphate (IP6) generated by IP5K mediates cullin-COP9 signalosome interactions and CRL function

doi: 10.1073/pnas.1525580113

Figure Lengend Snippet: The N-terminal acidic tail of CSN2 binds to the C-terminal basic canyons of cullins. (A) Pulldown of GST-CSN1/2/5/6 reveals coprecipitation of Cul1, -2, -3, and -4A with CSN2. (B) Direct interaction between E. coli-purified CSN2 and HEK293-purified GST-Cul4A. (C) Sequence the CSN2 N-terminal tail, the corresponding secondary structure was predicted by Psipred. Acidic residues in HsCSN2 are highlighted in red. (D) GST pulldown of CSN2 wild-type and its N-terminal deletion mutant Δ26. (E) The N-terminal tail of CSN2 is required for its ability to assemble CRL4A–CSN complexes. The shRNA targets the 3′-UTR of CSN2 and therefore does not influence transfected CSN2 (21). (F) Structure of Cul4A/Roc1 (PDB ID code 2HYE) shown in surface mode. The basic canyon is circled. (G) Mutations K465E and K675E abolish Cul4A binding to CSN2/5 but not to DDB1.

Article Snippet: The primary antibodies used were: Cul1, Cul2, HIF-1α, p21, p27 (Santa Cruz); Cul3 Cul4A, CSN5, K48-specific polyubiquitin, and DDB1 (Cell Signaling); GAPDH (Roche); IP5K and CSN2 (ProteinTech); NRF2 (R&D Systems); and GST (Sigma).

Techniques: Purification, Sequencing, Mutagenesis, shRNA, Transfection, Binding Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Inositol hexakisphosphate (IP6) generated by IP5K mediates cullin-COP9 signalosome interactions and CRL function

doi: 10.1073/pnas.1525580113

Figure Lengend Snippet: The N-terminal acidic tail of CSN2 binds to cullins. (A) Direct in vitro binding between CSN2 and GST-Cul4A wild-type, K705A, and K705R. (B) GST-Cul4A pulldown of recombinant CSN2 in the presence/absence of HA-Roc1. (C) High salt treatment markedly diminishes Cul4A binding to CSN subunits, with minimal effects on Cul4A–DDB1 interactions. (D) Sequence alignment of the CSN2 N-terminal tail, the corresponding secondary structure was predicted by Psipred. Acidic residues in HsCSN2 are highlighted in red. At, Arabidopsis thaliana; Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; Dr, Danio rerio; Gg, Gallus gallus; Hs, Homo sapiens; Mm, Mus musculus. (E) Deletion of the N-terminal 17, but not the N-terminal 8, residues abolishes binding of CSN2 to cullins. (F) Pulldown of CSN2 wild-type and various mutants. (G) Amino acids 1–40 of CSN2 are sufficient to bind to Cul1, -2, -3, -4A. After transfection of the myc-cullin constructs, recombinant GST-rCSN21–40 on beads was incubated with cell lysates. (H) Recombinant CSN2 Δ26 is not pulled down by purified GST-Cul4A in vitro.

Article Snippet: The primary antibodies used were: Cul1, Cul2, HIF-1α, p21, p27 (Santa Cruz); Cul3 Cul4A, CSN5, K48-specific polyubiquitin, and DDB1 (Cell Signaling); GAPDH (Roche); IP5K and CSN2 (ProteinTech); NRF2 (R&D Systems); and GST (Sigma).

Techniques: In Vitro, Binding Assay, Recombinant, Sequencing, Transfection, Construct, Incubation, Purification

Journal: Nature Communications

Article Title: The RepID–CRL4 ubiquitin ligase complex regulates metaphase to anaphase transition via BUB3 degradation

doi: 10.1038/s41467-019-13808-9

Figure Lengend Snippet: a RepID-CRL4 localizes with BUB3 at the kinetochore in prometaphase cells. Immunofluorescence analysis of HCT116 RepID WT and KO cells was carried out in methanol-fixed cells using antibodies directed against CRL4 components (RepID, CUL4A, CUL4B, or DDB1; red signals) and BUB3 (green signal). CRL4 components do not associate with chromatin in the RepID KO cells because RepID, which recruits CRL4 to chromatin, is absent. Scale bar: 10 μm. b RepID-dependent CRL4 recruitment to chromatin throughout the cell cycle. Mitotic HCT116 RepID WT or KO cells were collected after a nocodazole block, reseeded in drug-free medium, harvested at the indicated time points, and chromatin-bound proteins were analyzed by immunoblotting and densitometry. Histone H3 and α-Tubulin were used as a loading control. c , d BUB3 is degraded in mitosis in RepID-expressing cells but not in RepID-deficient cells. HCT116 RepID WT or KO cells were released from mitotic block for the indicated time periods with or without the proteasome inhibitor MG132. Cell lysates were subjected to immunoblot analysis and densitometry. e BUB3 is ubiquitinated during mitosis in RepID-expressing cells but not in RepID-deficient cells. HIS-tagged ubiquitin plasmids were transiently transfected into HCT116 RepID WT and KO cells. Cells were collected at the indicated times after release from a nocodazole block, and his-tagged proteins from the released cells were isolated on Ni-NTA beads followed by immunoblot analysis. f Knockdown of CUL4 prevented the ubiquitination of BUB3 in RepID-expressing cells. HIS-ubiquitin plasmid and siRNA-CUL4 (directed against both CUL4A and CUL4B) were co-transfected into HCT116 RepID WT cells, which were synchronized, released, lysed, isolated with Ni-NTA beads, and analyzed for the presence of ubiquitinated BUB3 by immunoblotting as in e .

Article Snippet: Human FLAG-tagged expression plasmids for CUL4A (RC214798), CUL4B (RC206935), DDB1 (RC208372), BUB3 (RC200376), and RBBP7 (RC231256) were purchased from ORIGENE.

Techniques: Immunofluorescence, Blocking Assay, Western Blot, Expressing, Transfection, Isolation, Plasmid Preparation

Journal: Nature Communications

Article Title: The RepID–CRL4 ubiquitin ligase complex regulates metaphase to anaphase transition via BUB3 degradation

doi: 10.1038/s41467-019-13808-9

Figure Lengend Snippet: a RepID dissociates from CRL4 during mitotic progression. HCT116 cells were transfected with RepID FL expression plasmid (for constructs, see Fig. ). Cells were collected at the indicated times after release from a nocodazole block, and chromatin-bound fractions were extracted. Immunoprecipitation was performed using anti-FLAG antibody, and co-precipitated proteins were analyzed by immunoblotting. b RBBP7 is incorporated into CRL4 after mitosis. Chromatin fractions of FLAG-DDB1-transfected HCT116 cells released from mitotic block at the indicated time points were immunoprecipitated with anti-FLAG antibody, followed by immunoblot analysis. c RBBP7, but not other DCAFs, localizes to mitotic spindle during mitosis. Immunofluorescence analysis was performed using the indicated anti-DCAFs antibodies in HCT116 WT cells. Scale bar: 10 μm. d RBBP7 colocalizes with BUB3 during metaphase. HCT116 RepID WT or KO cells were subject to immunofluorescence analysis using anti-RBBP7, BUB3, and anti-CREST antibodies with/without treatment with a p97/VCP inhibitor (CB5083). Zoomed-in areas (marked by white squares) are shown at the bottom of the composite images (merged images of the zoomed-in area are shown to the left of the single-color zoomed-in images). Scale bar: 10 μm. Quantification of colocalization between RBBP7 and BUB3 by Pearson’s correlation coefficients (bottom panel) (* p value < 0.05, ** p < 0.01, *** p < 0.001, n.s. not significant, Student’s t test). e Depletion of RepID-CRL4 RBBP7 components or BUB3 overexpression can delay mitotic exit. Depleted or overexpressed HCT116 cells were released from a nocodazole arrest for 3 h and analyzed by flow cytometry. Percentages of cells in subG1, G1, G2/M, and >4 N phase are indicated. f RBBP7 facilitates the ubiquitination of BUB3 during mitosis. HIS-ubiquitin plasmids were transfected into HCT116 RepID WT cells together with siRNA-RBBP4 or si-RBBP7. Cells were synchronized, released, lysed, and proteins isolated on Ni-NTA beads as described in the legend for Fig. . Immunoblot analysis was carried out using anti-BUB3 antibodies. g A model describing the DCAF switch on chromatin during mitosis. RepID recruits CRL4 on chromatin before metaphase, bringing CRL4 in contact with BUB3. RepID then dissociates from CRL4, and RBBP7 is incorporated into CRL4 as a catalytic DCAF for BUB3 ubiquitination.

Article Snippet: Human FLAG-tagged expression plasmids for CUL4A (RC214798), CUL4B (RC206935), DDB1 (RC208372), BUB3 (RC200376), and RBBP7 (RC231256) were purchased from ORIGENE.

Techniques: Transfection, Expressing, Plasmid Preparation, Construct, Blocking Assay, Immunoprecipitation, Western Blot, Immunofluorescence, Over Expression, Flow Cytometry, Isolation

Journal: The Journal of Biological Chemistry

Article Title: Amyloid Precursor Protein (APP) May Act as a Substrate and a Recognition Unit for CRL4 CRBN and Stub1 E3 Ligases Facilitating Ubiquitination of Proteins Involved in Presynaptic Functions and Neurodegeneration *

doi: 10.1074/jbc.M116.733626

Figure Lengend Snippet: E1, E2, and E3 interacting with the ACR, Ddb1, Crbn, Stub1, Cul4a, and Cul4b, are the most abundant UPS-linked proteins that bind to the ACR Table contains the list of proteins identified (1st column); the database accession numbers (2nd column); the molecular mass in kDa (3rd column); and the NSAF (4th to 8th columns. Phosphorylation of Thr 668 and/or Tyr 682 does not significantly alter binding of these five proteins.

Article Snippet: The following antibodies were used in WB: anti-Ddb1 (catalog no. 6998s, lot 1, rabbit monoclonal antibody, Cell Signaling Technology, dilution 1:300); anti-Pin-1 (catalog no. 3722s, lot 3, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:1000); anti-Stub1 (catalog no. 2080s, lot 2, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:300); anti-Grb2 (catalog no. 3972s, lot 2, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:1000); anti-Cul4a (catalog no. 14851-1-AP, lot 1, rabbit polyclonal antibody, ProteinTech, dilution 1:300); anti-Crbn (catalog no. HPA045910, lot Q103829, rabbit polyclonal antibody, Sigma, dilution 1:500); and anti-FLAG M2 (catalog no. A2220, lot SLBF8148, mouse monoclonal antibody, Sigma, dilution 1:1000).

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Amyloid Precursor Protein (APP) May Act as a Substrate and a Recognition Unit for CRL4 CRBN and Stub1 E3 Ligases Facilitating Ubiquitination of Proteins Involved in Presynaptic Functions and Neurodegeneration *

doi: 10.1074/jbc.M116.733626

Figure Lengend Snippet: Stub1 and CRL4CRBN E3 ligases bind distinct regions of the ACR. A, schematic diagrams of the structural domains of the ACR. Jcasp and Ccas are generated by a double γ-secretase and caspase cleavage of APP. The Thr668 and Tyr682 residues that are phosphorylated are underlined. B, Western blotting analysis of pulldowns shows that Crbn, Ddb1, Cul4a, specifically bind St-Ccas, St-CcasTyr(P), and St-CcasThr(P). Stub1 binds specifically St-JCasp. Two previously known APP interactors, Grb2 and Pin1, bind St-CcasTyr(P) and St-CcasThr(P), respectively. This evidence validates the proteomic approach used. In. indicates the input. The WB shown are representative of at least three independent experiments.

Article Snippet: The following antibodies were used in WB: anti-Ddb1 (catalog no. 6998s, lot 1, rabbit monoclonal antibody, Cell Signaling Technology, dilution 1:300); anti-Pin-1 (catalog no. 3722s, lot 3, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:1000); anti-Stub1 (catalog no. 2080s, lot 2, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:300); anti-Grb2 (catalog no. 3972s, lot 2, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:1000); anti-Cul4a (catalog no. 14851-1-AP, lot 1, rabbit polyclonal antibody, ProteinTech, dilution 1:300); anti-Crbn (catalog no. HPA045910, lot Q103829, rabbit polyclonal antibody, Sigma, dilution 1:500); and anti-FLAG M2 (catalog no. A2220, lot SLBF8148, mouse monoclonal antibody, Sigma, dilution 1:1000).

Techniques: Generated, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Amyloid Precursor Protein (APP) May Act as a Substrate and a Recognition Unit for CRL4 CRBN and Stub1 E3 Ligases Facilitating Ubiquitination of Proteins Involved in Presynaptic Functions and Neurodegeneration *

doi: 10.1074/jbc.M116.733626

Figure Lengend Snippet: Ddb1, Crbn, Cul4a, and Cul4b bind to Ccas and Stub1 binds to JCasp Phosphorylation of Thr 668 and/or Tyr 682 does not significantly alter binding of Ddb1, Crbn, Cul4a, and Cul4b, whereas binding of Pin1 and Grb2 is dependent on phosphorylation of Thr 668 and Tyr 682 , respectively. Several of the other UPS-related proteins that are bound to the ACR bind to either Ccas or JCasp or both.

Article Snippet: The following antibodies were used in WB: anti-Ddb1 (catalog no. 6998s, lot 1, rabbit monoclonal antibody, Cell Signaling Technology, dilution 1:300); anti-Pin-1 (catalog no. 3722s, lot 3, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:1000); anti-Stub1 (catalog no. 2080s, lot 2, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:300); anti-Grb2 (catalog no. 3972s, lot 2, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:1000); anti-Cul4a (catalog no. 14851-1-AP, lot 1, rabbit polyclonal antibody, ProteinTech, dilution 1:300); anti-Crbn (catalog no. HPA045910, lot Q103829, rabbit polyclonal antibody, Sigma, dilution 1:500); and anti-FLAG M2 (catalog no. A2220, lot SLBF8148, mouse monoclonal antibody, Sigma, dilution 1:1000).

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Amyloid Precursor Protein (APP) May Act as a Substrate and a Recognition Unit for CRL4 CRBN and Stub1 E3 Ligases Facilitating Ubiquitination of Proteins Involved in Presynaptic Functions and Neurodegeneration *

doi: 10.1074/jbc.M116.733626

Figure Lengend Snippet: Binding of Ddb1, Cul4a, and Cul4b to ACR Thr(P)Tyr(P) requires Crbn Table contains the list of proteins identified (1st column); the database accession numbers (2nd column); the molecular mass in kDa (3rd column); NSAF of pulldown from WT mouse brains (4th column); NSAF of pulldown from Crbn -KO mouse brains (5th column). Binding of Stub1, Grb2, and Pin1 is independent of Crbn.

Article Snippet: The following antibodies were used in WB: anti-Ddb1 (catalog no. 6998s, lot 1, rabbit monoclonal antibody, Cell Signaling Technology, dilution 1:300); anti-Pin-1 (catalog no. 3722s, lot 3, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:1000); anti-Stub1 (catalog no. 2080s, lot 2, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:300); anti-Grb2 (catalog no. 3972s, lot 2, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:1000); anti-Cul4a (catalog no. 14851-1-AP, lot 1, rabbit polyclonal antibody, ProteinTech, dilution 1:300); anti-Crbn (catalog no. HPA045910, lot Q103829, rabbit polyclonal antibody, Sigma, dilution 1:500); and anti-FLAG M2 (catalog no. A2220, lot SLBF8148, mouse monoclonal antibody, Sigma, dilution 1:1000).

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Amyloid Precursor Protein (APP) May Act as a Substrate and a Recognition Unit for CRL4 CRBN and Stub1 E3 Ligases Facilitating Ubiquitination of Proteins Involved in Presynaptic Functions and Neurodegeneration *

doi: 10.1074/jbc.M116.733626

Figure Lengend Snippet: Crbn mediates the interaction of CRL4CRBN with APP. Western blotting analysis shows that with St and St-ACRThr(P)Tyr(P) binds Ddb1 only when Crbn in present in brain lysates, indicating that Crbn mediates the binding of CRL4CRBN to the ACR. Binding of Grb2 and Pin1 to St-ACRThr(P)Tyr(P) is not dependent on Crbn. The WB shown is representative of 4 independent experiments.

Article Snippet: The following antibodies were used in WB: anti-Ddb1 (catalog no. 6998s, lot 1, rabbit monoclonal antibody, Cell Signaling Technology, dilution 1:300); anti-Pin-1 (catalog no. 3722s, lot 3, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:1000); anti-Stub1 (catalog no. 2080s, lot 2, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:300); anti-Grb2 (catalog no. 3972s, lot 2, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:1000); anti-Cul4a (catalog no. 14851-1-AP, lot 1, rabbit polyclonal antibody, ProteinTech, dilution 1:300); anti-Crbn (catalog no. HPA045910, lot Q103829, rabbit polyclonal antibody, Sigma, dilution 1:500); and anti-FLAG M2 (catalog no. A2220, lot SLBF8148, mouse monoclonal antibody, Sigma, dilution 1:1000).

Techniques: Western Blot, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Amyloid Precursor Protein (APP) May Act as a Substrate and a Recognition Unit for CRL4 CRBN and Stub1 E3 Ligases Facilitating Ubiquitination of Proteins Involved in Presynaptic Functions and Neurodegeneration *

doi: 10.1074/jbc.M116.733626

Figure Lengend Snippet: APP-CRL4CRBN complex is mediated by the interaction of the COOH terminus of APP with the substrate recognition pocket of Crbn. A, sequences of the APP intracellular regions used as baits (St-Ccas, St-C28, St-C24, St-C20, St-C16, St-C12, St-C8, and St-C4) in the proteomic experiments are shown. Western blotting analysis of pulldowns with brains isolated from WT mice show that the COOH-terminal 12 amino acids of APP retain full binding activity to Crbn and Ddb1. The WB shown is representative of two independent experiments. B, sequences of the APP intracellular regions used as baits (St-C12, St-C12δC1, St-C12δC2, St-C12δC3, St-C12δC5, and St- St-C12δC7) in the proteomic experiments are shown. The WB shown is representative of two independent experiments. C, Western blotting analysis shows that thalidomide (Thal.) and lenalidomide (Len.), two compounds that bind to the substrate recognition pocket of Crbn, reduce binding of Crbn and Ddb1 from brain lysates to St-CcasTyr(P) in a dose-dependent manner. Thalidomide and lenalidomide do not interfere with binding of Grb2 to St-CcasTyr(P). Because Pin1 binding is dependent on phosphorylation of Thr668 of APP, Pin1 does not bind the St-CcasTyr(P) bait and is used as a negative control to show binding specificity. The WB shown is representative of three independent experiments. D, schematic model representing how Crbn mediated binding of CRL4CRBN to the COOH terminus of APP, via its substrate recognition pocket.

Article Snippet: The following antibodies were used in WB: anti-Ddb1 (catalog no. 6998s, lot 1, rabbit monoclonal antibody, Cell Signaling Technology, dilution 1:300); anti-Pin-1 (catalog no. 3722s, lot 3, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:1000); anti-Stub1 (catalog no. 2080s, lot 2, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:300); anti-Grb2 (catalog no. 3972s, lot 2, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:1000); anti-Cul4a (catalog no. 14851-1-AP, lot 1, rabbit polyclonal antibody, ProteinTech, dilution 1:300); anti-Crbn (catalog no. HPA045910, lot Q103829, rabbit polyclonal antibody, Sigma, dilution 1:500); and anti-FLAG M2 (catalog no. A2220, lot SLBF8148, mouse monoclonal antibody, Sigma, dilution 1:1000).

Techniques: Western Blot, Isolation, Binding Assay, Activity Assay, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Amyloid Precursor Protein (APP) May Act as a Substrate and a Recognition Unit for CRL4 CRBN and Stub1 E3 Ligases Facilitating Ubiquitination of Proteins Involved in Presynaptic Functions and Neurodegeneration *

doi: 10.1074/jbc.M116.733626

Figure Lengend Snippet: AL2CR, but not the AL1CR, binds CRL4CRBN via the substrate recognition pocket of Crbn. A, alignments of the ACR, AL1CR, and AL2CR show that the ACR and the AL2CR are the most conserved intracellular regions of the APP protein family. B, Western blotting analysis of pulldowns with brains isolated from WT mice shows that the AL2CR, but not the AL1CR, interacts with Cul4a, Ddb1, and Crbn. The WB shown is representative of two independent experiments. C, St-AL2CR binds Ddb1 in lysate from WT but not from Crbn-KO mice. The WB shown is representative of three independent experiments. D, Western blotting analysis of St-AL2CR pulldowns from brains isolated from WT mice shows that incubation of the lysates with either thalidomide and lenalidomide prior to pulldowns interferes with the AL2CR-Crbn interaction. The WB shown is representative of two independent experiments.

Article Snippet: The following antibodies were used in WB: anti-Ddb1 (catalog no. 6998s, lot 1, rabbit monoclonal antibody, Cell Signaling Technology, dilution 1:300); anti-Pin-1 (catalog no. 3722s, lot 3, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:1000); anti-Stub1 (catalog no. 2080s, lot 2, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:300); anti-Grb2 (catalog no. 3972s, lot 2, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:1000); anti-Cul4a (catalog no. 14851-1-AP, lot 1, rabbit polyclonal antibody, ProteinTech, dilution 1:300); anti-Crbn (catalog no. HPA045910, lot Q103829, rabbit polyclonal antibody, Sigma, dilution 1:500); and anti-FLAG M2 (catalog no. A2220, lot SLBF8148, mouse monoclonal antibody, Sigma, dilution 1:1000).

Techniques: Western Blot, Isolation, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Amyloid Precursor Protein (APP) May Act as a Substrate and a Recognition Unit for CRL4 CRBN and Stub1 E3 Ligases Facilitating Ubiquitination of Proteins Involved in Presynaptic Functions and Neurodegeneration *

doi: 10.1074/jbc.M116.733626

Figure Lengend Snippet: UPS-related proteins, including Ddb1, Crbn, Stub1, Cul4a, and Cul4b, bind to the AL2CR but not to the AL1CR The table contains the list of proteins identified (1st column); the database accession numbers (2nd column); the molecular mass in kDa (3rd column); NSAF of pulldown of WT mouse brains with St alone (4th column); St-AL1CR (5th column); and NSAF of pulldown of WT mouse brains with St-AL2CR (6th column).

Article Snippet: The following antibodies were used in WB: anti-Ddb1 (catalog no. 6998s, lot 1, rabbit monoclonal antibody, Cell Signaling Technology, dilution 1:300); anti-Pin-1 (catalog no. 3722s, lot 3, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:1000); anti-Stub1 (catalog no. 2080s, lot 2, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:300); anti-Grb2 (catalog no. 3972s, lot 2, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:1000); anti-Cul4a (catalog no. 14851-1-AP, lot 1, rabbit polyclonal antibody, ProteinTech, dilution 1:300); anti-Crbn (catalog no. HPA045910, lot Q103829, rabbit polyclonal antibody, Sigma, dilution 1:500); and anti-FLAG M2 (catalog no. A2220, lot SLBF8148, mouse monoclonal antibody, Sigma, dilution 1:1000).

Techniques: