Journal: Science advances

Article Title: A cancer ubiquitome landscape identifies metabolic reprogramming as target of Parkin tumor suppression.

doi: 10.1126/sciadv.abg7287

Figure Lengend Snippet: Fig. 3. Landscape of a Parkin proteome and ubiquitome in cancer. (A) Scatter plots of protein intensities from two independent global proteome SILAC experiments in PC3 cells transfected with vector (light labeled) or Parkin (heavy labeled) in the absence of mitochondrial uncouplers. A Pearson correlation coefficient is indicated. (B) The conditions are as in (A), and concordance in detection of proteins between experiment 1 (Exp. 1, 97%) and experiment 2 (Exp. 2, 96%) is shown. (C) Changes in protein levels in PC3 cells transfected with Parkin. A 1.3-fold difference cutoff was defined as potentially biologically important. (D) STRING (Search Tool for the Retrieval of Inter- acting Genes/Proteins) analysis of predicted protein networks modulated by Parkin in the global proteome of PC3 cells. (E) Volcano plot of the Parkin global proteome in PC3 cells. The distribution of total proteins (N = 5557) and MitoCarta 3.0–associated proteins (N = 741) is indicated. (F) STRING analysis of predicted mitochondrial protein networks modulated by Parkin in the global proteome. (G) Volcano plot of a Parkin ubiquitome in PC3 cells in the absence of mitochondrial uncouplers. The distribution of total (N = 3796) and MitoCarta 3.0–associated ubiquitination sites (N = 112) is indicated. (H) Changes in Parkin ubiquitome identified using SILAC-based ubiquitin rem- nant motif enrichment (K--GG), with 1013 and 423 sites showing increased or decreased ubiquitination in the presence of Parkin using a 1.3-fold cutoff.

Article Snippet: SILAC ubiquitome and global proteomic analyses PC3 cells expressing Parkin (heavy SILAC labeled with 13C615N4 l-arginine, 13C615N2 l-lysine) or vector (light SILAC labeled) were processed in duplicate for ubiquitome analysis using the PTMScan Ubiquitin Remnant Motif (K--GG) Kit (Cell Signaling Technology).

Techniques: Multiplex sample analysis, Transfection, Plasmid Preparation, Labeling, Ubiquitin Proteomics

Journal: Science advances

Article Title: A cancer ubiquitome landscape identifies metabolic reprogramming as target of Parkin tumor suppression.

doi: 10.1126/sciadv.abg7287

Figure Lengend Snippet: Fig. 4. Novel Parkin ubiquitome in cancer. (A) Bioinformatics analysis of predicted protein networks regulated by Parkin ubiquitination in the absence of mitochondrial uncoupling, including cell death (HK1, MCL1, and HMGB1), glucose metabolism involving glycolysis (HK1 and TPI1) and the PPP (TALDO1 and TKT), protein folding (CCT7 and HSPA1A, also known as HSP72), and mitochondrial dynamics (RHOT1, FIS1, and MFN2). Parkin-directed ubiquitination sites (Lys, K) identified by SILAC proteomics in each target protein are indicated. (B) PC3 cells in the presence or absence of Parkin were immunoprecipitated with an antibody to MFN2, and immune complexes were probed with antibodies to ubiquitin (Ub) or MFN2 by Western blotting. IP, immunoprecipitation; IB, immunoblot. (C) The conditions are as in (B) except that TKT immune complexes precipitated from PC3 cells with or without Parkin were probed with antibodies to Parkin, TKT or Ub, by Western blotting. (D) PC3 cells transfected with WT Parkin or E3 ligase–defective Parkin S65A mutant were analyzed by Western blotting. (E) PC3 cells transfected with vector (Veh) or Parkin (P) were treated with bafilomycin A (BafA; 20 nM for 6 hours) and analyzed by Western blotting. (F) PC3 cells transfected with vector or Parkin were treated with the indicated concentrations of BafA and analyzed by Western blotting. (G) PC3 cells transfected with vector or Parkin were analyzed for changes in mRNA expression of RHOT1, TKT, or TALDO1 by reverse transcrip- tion PCR. Mean ± SD (N = 3). (H) The indicated tumor cell types transfected as in (E) were analyzed by Western blotting. (I) Comparison between the Parkin ubiquitination sites identified in PC3 cells in the absence of mitochondrial uncoupling (this study) and HeLa cells treated with CCCP plus Velcade (Exp. ID 57) as reported in (27). The num- ber of ubiquitination sites is indicated. Only sites showing significant changes in SILAC ratios as defined in the respective studies were compared.

Article Snippet: SILAC ubiquitome and global proteomic analyses PC3 cells expressing Parkin (heavy SILAC labeled with 13C615N4 l-arginine, 13C615N2 l-lysine) or vector (light SILAC labeled) were processed in duplicate for ubiquitome analysis using the PTMScan Ubiquitin Remnant Motif (K--GG) Kit (Cell Signaling Technology).

Techniques: Ubiquitin Proteomics, Multiplex sample analysis, Immunoprecipitation, Western Blot, Transfection, Mutagenesis, Plasmid Preparation, Expressing, Comparison

Journal: Communications Biology

Article Title: Alteration in tyrosine phosphorylation of cardiac proteome and EGFR pathway contribute to hypertrophic cardiomyopathy

doi: 10.1038/s42003-022-04021-4

Figure Lengend Snippet: a Myofilament from Ntg ( n = 3) and ErbB2 ( n = 3) transgenic mice hearts were freshly isolated on ice-cold buffers containing Proteinase and Phosphatase Inhibitors Cocktails (Roche). b All material was resuspended in TEAB, then reduced and alkylated. Tryptic peptides were desalted and labeled with 6-plex isobaric tandem mass tags (TMT). c The digested and labeled peptides were pooled and desalted with C 18 SEP-PAK. The enrichment for phosphotyrosine was performed with PTMScan Phospho-Tyrosine Rabbit mAb ( P -Tyr-1000) kit (Cell Signaling Technology). The eluted peptide samples were desalted using C18 STAGE tips d Easy-nanoLC 1200 nanoflow liquid chromatography system coupled to Orbitrap Fusion Lumos Tribrid.

Article Snippet: The digested and labeled peptides were pooled and desalted with C 18 SEP-PAK (Waters), followed by pTyrsoine enrichment using PTMScan® Phospho-Tyrosine Rabbit mAb (P-Tyr-1000) kit (Cell Signaling Technology), see Fig. .

Techniques: Transgenic Assay, Isolation, Labeling, Liquid Chromatography