Dataset Information

Global redox proteome and phosphoproteome analysis reveals novel mode of Akt regulation

ABSTRACT: A major knowledge gap in cell signalling is defining the crosstalk between multiple types of post-translational modification (PTM). Here, we take a multi-omic approach (redox proteome, phosphoproteome and total proteome) to delineate signalling networks in adipocytes modulated by reactive oxygen species (ROS) and protein phosphorylation. Our integrative analysis of these datasets revealed widespread and complex crosstalk between oxidative stress-induced cysteine oxidation and phosphorylation-based signalling. In particular, we demonstrate dysregulation in the kinase substrate relationships of Akt, mTOR and AMPK. Furthermore, we identify that Cys60 and Cys77 in the pleckstrin homology (PH) domain of Akt are required for its recruitment to the plasma membrane, its subsequent activation, and its regulation by redox. These multi-omics datasets provide insight into how redox signalling driven by oxidative stress interacts with protein phosphorylation and should serve as a useful resource for dissecting oxidative stress-induced PTMs and understanding their contribution to a variety of diseases.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Adipocyte Cell Line, Preadipocyte

DISEASE(S): Type 2 Diabetes Mellitus

SUBMITTER: Zhiduan Su

LAB HEAD: David E. James

PROVIDER: PXD011525 | Pride | 2019-12-10

REPOSITORIES: Pride

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Publications

Global redox proteome and phosphoproteome analysis reveals redox switch in Akt.

Su Zhiduan Z Burchfield James G JG Yang Pengyi P Humphrey Sean J SJ Yang Guang G Francis Deanne D Yasmin Sabina S Shin Sung-Young SY Norris Dougall M DM Kearney Alison L AL Astore Miro A MA Scavuzzo Jonathan J Fisher-Wellman Kelsey H KH Wang Qiao-Ping QP Parker Benjamin L BL Neely G Gregory GG Vafaee Fatemeh F Chiu Joyce J Yeo Reichelle R Hogg Philip J PJ Fazakerley Daniel J DJ Nguyen Lan K LK Kuyucak Serdar S James David E DE

Nature communications 20191202 1

Protein oxidation sits at the intersection of multiple signalling pathways, yet the magnitude and extent of crosstalk between oxidation and other post-translational modifications remains unclear. Here, we delineate global changes in adipocyte signalling networks following acute oxidative stress and reveal considerable crosstalk between cysteine oxidation and phosphorylation-based signalling. Oxidation of key regulatory kinases, including Akt, mTOR and AMPK influences the fidelity rather than the ...[more]

PMID: 31792197

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Project description:Animal mRNAs are regulated by hundreds of RNA binding proteins (RBPs). The identification of RBP targets is crucial for understanding their function. A recent method, PAR-CLIP, uses photoreactive nucleosides to crosslink RBPs to target RNAs in cells prior to immunoprecipitation. Here, we establish iPAR-CLIP (in vivo PAR-CLIP) to determine, at nucleotide resolution, transcriptome-wide target sites of GLD-1, a conserved, germline-specific translational repressor in C. elegans. We identified 439 reproducible targets and demonstrate an excellent dynamic range of target detection by iPAR-CLIP. Upon GLD-1 knock-down, protein but not mRNA expression of the 439 targets was specifically and highly significantly upregulated, demonstrating functionality. Finally, we discovered strongly conserved GLD-1 binding sites nearby the start codon of target genes. We propose that GLD-1 interacts with the translation machinery nearby the start codon, a so far unknown mode of gene regulation in eukaryotes. Arrested L1 worms were grown in liquid culture supplemented with 2mM 4SU or 6SG. 250,000 worms were sufficient for one iPAR-CLIP experiment. Living adult worms were transferred to NGM plates and crosslinked on ice using a Stratalinker (Stratagene) with customized 365nm UV-lamps (energy setting: 2J/cm2). Worms were lysed on ice by douncing in NP40 lysis buffer (50 mM HEPES-K pH 7.5, 150 mM KCl, 2 mM EDTA, 0.5% (v/v) NP-40, 0.5 mM DTT, protease inhibitor cocktail (Roche)). Cleared lysates were treated with RNase T1 (Fermentas) (final concentration 1U/?l) for 15 min at 22ÂºC. GLD-1::GFP::FLAG fusion proteins were immunoprecipitated for 1h at 4ÂºC using anti-FLAG antibody (Sigma, F3165) coupled to Protein G magnetic beads (Invitrogen). For one iPAR-CLIP experiment (1ml cleared lysate obtained from 250,000 worms), 300Âµl beads and 150Âµg antibody were used. Immunoprecipitates were treated with RNase T1 (100U/?l) for exactly 12 min at 22 ÂºC. Subsequently, PAR-CLIP was carried out as described previously (Hafner et al, 2010). cDNA libraries were sequenced on a Genome Analyzer II (Illumina).

Dataset Information

Global redox proteome and phosphoproteome analysis reveals novel mode of Akt regulation

Publications

Global redox proteome and phosphoproteome analysis reveals redox switch in Akt.

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