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LFQ of biotinylated peptides from HEK293T cells expressing AirID-CRBN

ABSTRACT: HEK293T cells stably expressing AGIA-AirID-CRBN-WT were treated with DMSO, 20 µM thalidomide, 10 µM lenalidomide, 10 µM pomalidomide, or 20 µM 5-hydroxythalidomide (5HT) in the presence of 10 µM biotin and 5 µM MG132 for 8 h in three biological replicates. After tryptic digestion, biotinylated peptides were enriched with Tamavidin 2-REV and analyzed by LC-MS/MS.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Hidetaka Kosako

PROVIDER: PXD028756 | JPOST Repository | Sat Nov 06 00:00:00 GMT 2021

REPOSITORIES: jPOST

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A proximity biotinylation-based approach to identify protein-E3 ligase interactions induced by PROTACs and molecular glues.

Yamanaka Satoshi S Horiuchi Yuto Y Matsuoka Saya S Kido Kohki K Nishino Kohei K Maeno Mayaka M Shibata Norio N Kosako Hidetaka H Sawasaki Tatsuya T

Nature communications 20220110 1

Proteolysis-targeting chimaeras (PROTACs) as well as molecular glues such as immunomodulatory drugs (IMiDs) and indisulam are drugs that induce interactions between substrate proteins and an E3 ubiquitin ligases for targeted protein degradation. Here, we develop a workflow based on proximity-dependent biotinylation by AirID to identify drug-induced neo-substrates of the E3 ligase cereblon (CRBN). Using AirID-CRBN, we detect IMiD-dependent biotinylation of CRBN neo-substrates in vitro and identif ...[more]

PMID: 35013300

Similar Datasets

Neo-substrate selectivity of IMiDs on biotinylation by AirID-CRBN

Project description:MM1.S cells stably expressing AGIA-AirID-CRBN-WT were treated with DMSO, 20 µM thalidomide, 10 µM lenalidomide, 10 µM pomalidomide or 20 µM 5-hydroxythalidomide (5HT) in the presence of 10 µM biotin and 5 µM MG132 for 8 h in three biological replicates. After tryptic digestion, biotinylated peptides were enriched with Tamavidin 2-REV and analysed by LC-MS/MS.

2021-11-06 | PXD028755 | JPOST Repository

LFQ of biotinylated peptides from IMR32 cells expressing AirID-CRBN

Project description:IMR32 cells stably expressing AGIA-AirID-CRBN-WT were treated with DMSO, 20 µM thalidomide, 10 µM lenalidomide, 10 µM pomalidomide, or 20 µM 5-hydroxythalidomide (5HT) in the presence of 10 µM biotin and 5 µM MG132 for 8 h in three biological replicates. After tryptic digestion, biotinylated peptides were enriched with Tamavidin 2-REV and analyzed by LC-MS/MS.

2021-11-06 | PXD028758 | JPOST Repository

LFQ of biotinylated peptide from HuH7 cells expressing AirID-CRBN

Project description:HuH7 cells stably expressing AGIA-AirID-CRBN-WT were treated with DMSO, 20 µM thalidomide, 10 µM lenalidomide, 10 µM pomalidomide, or 20 µM 5-hydroxythalidomide (5HT) in the presence of 10 µM biotin and 5 µM MG132 for 8 h in three biological replicates. After tryptic digestion, biotinylated peptides were enriched with Tamavidin 2-REV and analyzed by LC-MS/MS.

2021-11-06 | PXD028757 | JPOST Repository

LFQ of biotinylated peptides from THP-1 cells expressing AirID-CRBN

Project description:THP-1 cells stably expressing AGIA-AirID-CRBN-WT were treated with DMSO, 20 µM thalidomide, or 10 µM pomalidomide in the presence of 10 µM biotin and 5 µM MG132 for 8 h in three biological replicates. After tryptic digestion, biotinylated peptides were enriched with Tamavidin 2-REV and analyzed by LC-MS/MS.

2021-11-06 | PXD028760 | JPOST Repository

LFQ of biotinylated peptides from IMR32 cells expressing AirID-CRBN or TurboID-CRBN

Project description:IMR32 cells stably expressing AGIA-AirID-CRBN or AGIA-TurboID-CRBN were treated with DMSO or 10 µM pomalidomide in the presence of 10 µM biotin and 5 µM MG132 for 2 h in three biological replicates. After tryptic digestion, biotinylated peptides were enriched with Tamavidin 2-REV and analyzed by LC-MS/MS analysis.

2021-11-06 | PXD028761 | JPOST Repository

CRL4(CRBN) ubiquitin ligase profiling

Project description:In the 1950s the drug thalidomide administered as a sedative to pregnant women led ot the birth of thousands of children with multiple defects. Despite its teratogenicity, thalidomide and ist IMiD derivatives recently emerged as effective treatments for multiple myeloma and 5q-dysplasia. IMiDs target the CUL4-RBX1-DDB1-CRBN (CRL4(CRBN)) ubiquitin ligase. Through an unbiased screen we identify the homeobox trranscription factor MEIS2 as an endogenous substrate of CRL4(CRBN). By definition, a specific target of CRL4(CRBN) is expected to have a very low intensity on negative control arrays (E1_E2), (E1_CRBN), (E1_E2_Cdt2), (E1_E2_Cdt2_revlimid), (E1_E2_Cdt2_CSN) or with CRL4(CRBN) in presence of inhibitor (E1_E2_CRBN_revlimid) and high intensity on arrays with CRL4(CRBN) (E1_E2_CRBN) or CRL4(CRBN) in presence of CSN (E1_E2_CRBN_CSN) Accordingly 16 protein microarrays were subjected to in vitro ubiquitylation using the following enzyme combinations: 3x Uba1+UbcH5a; 2x Uba1+UbcH5a+CRL4(DDB2); 3x Uba1+UbcH5a+CRL4(CRBN); 2x Uba1+UbcH5a+CRL4(CRBN)+lenalidomide; 2x Uba1+UbcH5a+CRL4(CRBN)+CSN; 2x Uba1+UbcH5a+CRL4(Cdt2); 1x Uba1+UbcH5a+CRL4(Cdt2)+CSN; 1x Uba1+UbcH5a+CRL4(Cdt2)+lenalidomide

2014-05-13 | E-GEOD-57554 | biostudies-arrayexpress

CRL4(CRBN) ubiquitin ligase profiling

2014-05-13 | GSE57554 | GEO

LFQ of PROTAC-dependent biotinylated peptides in MM1.S cells expressing AirID-CRBN

Project description:MM1.S cells stably expressing AGIA-AirID-CRBN-WT were treated with DMSO, 1 µM pomalidomide, or 0.1 µM ARV-825 in the presence of 5 µM biotin and 5 µM MG132 for 6 h in three biological replicates. After tryptic digestion, biotinylated peptides were enriched with Tamavidin 2-REV and analyzed by LC-MS/MS.

2021-11-06 | PXD028763 | JPOST Repository

Comparison of three enrichment methods of biotinylated peptides or proteins from AirID-CRBN-expressing MM1.S cells

Project description:MM1.S cells stably expressing AGIA-AirID-CRBN were treated with DMSO or 10 µM pomalidomide in the presence of 10 µM biotin and 5 µM MG132 for 8 h in three biological replicates. The cells were divided into three tubes and lysed. After tryptic digestion, biotinylated peptides were enriched using Tamavidin 2-REV magnetic beads or anti-biotin antibody beads. Biotinylated proteins were enriched using NanoLink streptavidin magnetic beads followed by digestion with Trypsin. The resultant peptides were analyzed by LC-MS/MS.

2021-11-06 | PXD028754 | JPOST Repository

Cereblon expression is required for the anti-myeloma activity of lenalidomide and pomalidomide [expression profiling]

Project description:The precise molecular mechanism of action and targets through which thalidomide and related immunomodulatory drugs (IMiDs) exert their anti-tumor effects remains unclear. We investigated the role of cereblon (CRBN), a primary teratogenic target of thalidomide, in the anti-myeloma activity of IMiDs. CRBN depletion is initially cytotoxic to human myeloma cells but surviving cells with stable CRBN depletion become highly resistant to both lenalidomide and pomalidomide, but not to the unrelated drugs bortezomib, dexamethasone and melphalan. Acquired deletion of CRBN was found to be the primary genetic event differentiating isogenic MM1.S cell lines cultured to be sensitive or resistant to lenalidomide and pomalidomide. Gene expression changes induced by lenalidomide were dramatically suppressed in the presence of CRBN depletion further demonstrating that CRBN is required for lenalidomide activity. Downstream targets of CRBN include interferon regulatory factor 4 (IRF4) previously reported to also be a target of lenalidomide. Patients exposed to and putatively resistant to lenalidomide had lower CRBN levels in paired samples before and after therapy. In summary, CRBN is an essential requirement for IMiD activity, and a possible biomarker for the clinical assessment of anti-myeloma efficacy. We included 15 samples from multiple myeloma cell lines.

2011-09-30 | E-GEOD-31421 | biostudies-arrayexpress

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