Dataset Information

ABIN1 is an signal-induced autophagy adaptor counteracting LUBAC-mediated linear ubiquitination

ABSTRACT: ABIN1 KO RAW264.7 cells and stably reconstituted with 3×FLAG-ABIN1 WT or D485N were subjected to mass spectrometric analysis. Three biological replicates were prepared for each condition. Cells were stimulated for 3 hours with 100 ng/ml LPS. ABIN1 was immunoprecipitated with 30 uL anti-FLAG M2 Magnetic Beads (M8823, Sigma-Aldrich) for 2 h at 4℃. Immunoprecipitated sample was eluted by incubation at 90°C for 5 min in 100 uL PTS buffer. Each sample was split into 3 tubes for protein digestion with Lys-C and trypsin, chymotrypsin, or Glu-C.

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Kazuhiro Iwai

PROVIDER: PXD024864 | JPOST Repository | Thu Feb 17 00:00:00 GMT 2022

REPOSITORIES: jPOST

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Publications

ABIN1 is a signal-induced autophagy receptor that attenuates NF-κB activation by recognizing linear ubiquitin chains.

Shinkawa Yutaka Y Imami Koshi K Fuseya Yasuhiro Y Sasaki Katsuhiro K Ohmura Koichiro K Ishihama Yasushi Y Morinobu Akio A Iwai Kazuhiro K

FEBS letters 20220304 9

Linear ubiquitin chains play pivotal roles in immune signaling by augmenting NF-κB activation and suppressing programmed cell death induced by various stimuli. A20-binding inhibitor of NF-κB 1 (ABIN1) binds to linear ubiquitin chains and attenuates NF-κB activation and cell death induction. Although interactions with linear ubiquitin chains are thought to play a role in ABIN1-mediated suppression of NF-κB and cell death, the underlying molecular mechanisms remain unclear. Here, we show that upon ...[more]

PMID: 35213742

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Project description:Purpose: The goals of this study are to compare NGS-derived transcriptome profiling (RNA-seq) of U251 cells with endogenous Gcn5 depletion and reconstituted expression of WT Flag-rGcn5 or Flag-rGcn5 Y645A / endogenous DLST depletion and reconstituted expression of WT V5-rDLST or V5-rDLST R224A/K226E Transcriptomes. And quantitatively analyze the cell signaling pathways that regulated by the studied mutants. Methods: total mRNA was extracted from U251 cells with endogenous Gcn5 depletion and reconstituted expression of WT Flag-rGcn5 or Flag-rGcn5 Y645A / endogenous DLST depletion and reconstituted expression of WT V5-rDLST or V5-rDLST R224A/K226E.llumina compatible libraries were prepared by using the KAPA Stranded mRNA-Seq Library Preparation Kit for Illumina® platforms (KAPA Biosystems). In brief, 250 ng of total RNA was enriched for poly-A-tailed mRNA by using Kapa’s mRNA Capture beads. Poly-A-enriched RNA was fragmented to a median size of 150 bp by using chemical fragmentation and converted into double-stranded cDNA with dUTP incorporated into the second cDNA strand. The ends of the double-stranded cDNA were polished, 5’-phosphorylated, and 3’-A-tailed for the ligation of indexed adapters. Adapter-ligated DNA fragments were amplified by 8 cycles of PCR. The strand with incorporated dUTP was not amplified. The resulting libraries were quantified by qPCR and assessed for size distribution by using the 4200 TapeStation (Agilent Technologies), and then multiplexed, 4 per pool, and sequenced on the Illumina’s NextSeq500 by using the mid-output, 75-bp paired-end read format. Results: Using an optimized data analysis workflow, we mapped about 50 million sequence reads per sample to the human genome and quantified the expression of protein coding genes in the U251 cells of WT Gcn5 and Gcn5 Y645A mutant/WT DLST and DLST R224A/K226E mutant. Altered expression of 3 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Conclusions: Our study represents the first detailed analysis of U251 cells with endogenous Gcn5 depletion and reconstituted expression of WT Flag-rGcn5 or Flag-rGcn5 Y645A / endogenous DLST depletion and reconstituted expression of WT V5-rDLST or V5-rDLST R224A/K226E Transcriptomes. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that Gcn5 Y45A mutant and DLST R224A/K226E mutant, which reduce histone H3 K79 succinylation, can regulate several cell signaling pathways that important for cancer cell proliferation.

Dataset Information

ABIN1 is an signal-induced autophagy adaptor counteracting LUBAC-mediated linear ubiquitination

Publications

ABIN1 is a signal-induced autophagy receptor that attenuates NF-κB activation by recognizing linear ubiquitin chains.

Similar Datasets

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